JP2018505681A - Multispecific antibody having affinity for human A33 antigen and DOTA metal complex and use thereof - Google Patents

Abstract

Described herein are multispecific binding agents that bind A33 and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid. Also provided herein is a method of using a multispecific binding agent or composition thereof for the detection, prevention and / or therapeutic treatment of diseases characterized by expression of A33 glycoprotein antigens, particularly colorectal cancer. Provided.

Description

This application claims priority with respect to US Provisional Patent Application 62 / 113,988, filed February 9, 2015, the contents of which are hereby incorporated by reference in their entirety.

  Antibody-based therapeutics are particularly promising in the treatment of cancer. Various formats (including monoclonal, murine, chimeric, humanized, human, full-length, Fab, PEGylated, radiolabeled, factor complexed, multispecific, etc.) Is being used. Over 30 therapeutic antibody factors with marketing authorization in the US or Europe (see: Reichert, mAbs 4: 3, 413, May / June 2012, which is hereby incorporated by reference in its entirety) Of these, two bispecific antibodies (Catumaxomab and Blinatumomab) produced using separate techniques have been approved for human use. Nevertheless, there is a need for the development of excellent and effective antibodies.

The present invention provides inter alia multispecific binding agents, which contain binding moieties that interact with specific targets. In many embodiments, such binding moiety is an antibody component or comprises an antibody component. In some embodiments, a multispecific binding agent of the invention comprises a binding element of humanized antibody A33 (referred to herein as huA33). In some embodiments, a multispecific binding agent of the invention comprises a first binding moiety based on huA33 and a second binding moiety that interacts with an organic or inorganic compound. Such donors have improved functional characteristics compared to parental binding agents lacking such components described herein.
In particular, the present invention provides improved multispecific (eg, bispecific) antibody agents, which include A33 glycoprotein and benzyl-1,4,7,10-tetraazacyclododecane-1,4, Combines with 7,10-tetraacetic acid (DOTA-Bn). Wherein the multispecific binding agent comprises one or more structural features of humanized antibody A33 (eg, one or more CDRs) and one or more features of monoclonal antibody 2D12.5 (eg, one or more CDRs). )including. In some embodiments, provided multispecific binding agents exhibit high tumor uptake and low toxicity to normal tissues (eg, bone marrow and kidney) compared to parent antibodies A33 and 2D12.5. In some embodiments, the provided multispecific binding agent overcomes sub-optimal tumor dose and therapeutic index deficiencies when used in a pre-targeted radioimmunotherapy approach to improve A33 positive tumors To do.
In some embodiments, the invention includes bispecificity comprising a first antigen binding site based on humanized antibody A33 (huA33) and a second antigen binding site based on monoclonal antibody 2D12.5. An antibody is provided. In some embodiments, the 2D12.5 antibody is humanized.
In some embodiments, the first antigen binding site and / or the second antigen binding site is one or more polypeptide chains or comprises a polypeptide chain.

In some embodiments, the one or more polypeptide chains comprise a heavy chain CDR found in the sequence set forth in Table 8. In some certain embodiments, the heavy chain CDRs are found with a humanized A33 antibody (huA33). In some embodiments, the one or more polypeptide chains comprise a light chain CDR found in the sequence set forth in Table 8. In some certain embodiments, the light chain CDRs are found with a humanized A33 antibody (huA33). In some embodiments, the one or more polypeptide chains comprise heavy and light chain CDRs found in one or more sequences listed in Table 8. In some certain embodiments, the heavy and light chain CDRs are found with a humanized A33 antibody (huA33).
In some embodiments, the first and / or second antigen binding site is or comprises a single chain variable fragment (scFv). In some embodiments, the first antigen binding site is comprised of an immunoglobulin molecule and the second antigen binding site is comprised of an scFv, scFab, Fab or Fv. In some embodiments, the second antigen binding site is scFv. In some certain embodiments, the second antigen binding site is C825 scFv. In some embodiments, the C825 scFv is humanized. In some embodiments, the scFv is linked to the C-terminus of the heavy chain of an immunoglobulin molecule. In some embodiments, the scFv is linked to the C-terminus of the light chain of the immunoglobulin molecule.
In some embodiments, the present invention provides a bispecific antibody, said bispecific antibody comprising an immunoglobulin molecule based on humanized antibody A33 (huA33) and benzyl-1,4,7, It is composed of scFv that binds to 10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA-Bn), where scFv is based on C825 and linked to the C-terminus of the light chain of huA33 Furthermore, the bispecific antibody is characterized by a limited immunological effect when administered to an organism.
In some embodiments, the immunoglobulin molecule is IgG. In some embodiments, the immunoglobulin molecule is a non-glycosylated IgG. In some embodiments, the immunoglobulin molecule is an IgG with a K322A substitution. In some embodiments, the immunoglobulin molecule is an aglycosylated IgG having a K322 substitution.

In various embodiments, the bispecific antibody of the invention comprises SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In various embodiments, the bispecific antibody of the invention comprises SEQ ID NO: 6 or SEQ ID NO: 7. In various embodiments, the bispecific antibody of the invention comprises SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, and further comprises SEQ ID NO: 6 or SEQ ID NO: 7.
In various embodiments, the bispecific antibody of the invention comprises SEQ ID NO: 2 and SEQ ID NO: 6, SEQ ID NO: 2 and SEQ ID NO: 7, SEQ ID NO: 3 and SEQ ID NO: 6, SEQ ID NO: 3. And SEQ ID NO: 7, SEQ ID NO: 4 and SEQ ID NO: 6, or SEQ ID NO: 4 and SEQ ID NO: 7.
In some embodiments, the invention provides an isolated nucleic acid, which includes a coding sequence for part or all of the polypeptide chain of a bispecific antibody described herein. In some certain embodiments, the coding sequence is codon optimized.
In some embodiments, the present invention provides an expression vector, which comprises a nucleic acid molecule described herein.
In some embodiments, the present invention provides a host cell, which comprises a host cell as described herein.
In some embodiments, the invention provides a method of making a bispecific antibody described herein, wherein the method is performed in a culture medium under conditions that allow expression of the bispecific antibody. Culturing the host cells described herein, and separating the bispecific antibody from the culture medium.
In some embodiments, the present invention provides compositions comprising the bispecific antibodies described herein.
In some embodiments, the present invention provides a pharmaceutical composition comprising a composition described herein or a bispecific antibody described herein.
In some embodiments, the present invention provides the use of a pharmaceutical composition described herein or a composition described herein for the treatment or diagnosis of cancer.
In some embodiments, the present invention provides the use of the bispecific antibodies, compositions or pharmaceutical compositions described herein for the treatment or detection of symptoms associated with A33 expression.

In some embodiments, the present invention provides kits comprising the bispecific antibodies described herein.
In some embodiments, the present invention provides the use of the bispecific antibodies described herein in the manufacture of a medicament for use in medicine.
In some embodiments, the present invention provides the use of the bispecific antibodies described herein in the manufacture of a medicament for use in a diagnostic test or assay.
In some embodiments, the present invention provides the use of the bispecific antibodies described herein in the manufacture of a medicament for the diagnosis of cancer.
In some embodiments, the present invention provides the use of the bispecific antibodies described herein in the manufacture of a medicament for the treatment of cancer.
In some embodiments, the present invention provides the use of the bispecific antibodies described herein in the manufacture of a medicament for the treatment of colorectal cancer, gastric cancer or pancreatic cancer.
In some embodiments, the present invention provides a method of treating a medical condition in a subject animal, wherein the medical condition is characterized by A33 expression, the method comprising: Administering a therapeutically effective amount of the bispecific antibody described in the document. In some embodiments, the medical condition includes an A33 positive tumor. In some certain embodiments, the medical condition is colorectal cancer, gastric cancer or pancreatic cancer.
In some embodiments, the present invention provides a method of killing tumor cells. The method includes the step of contacting tumor cells with a bispecific antibody, the bispecific antibody comprising a first antigen binding site based on humanized antibody A33 (huA33) and benzyl-1,4 , 7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA-Bn) is composed of a second antigen-binding site, which contact is observed to kill tumor cells And sufficient time.

In some embodiments, the invention provides a method of inhibiting tumor growth, the method comprising contacting a tumor cell with a bispecific antibody, wherein the bispecific antibody is a humanized antibody. From the first antigen-binding site based on A33 and the second antigen-binding site that binds to benzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) Configured, the contact is such that unbound (unbound) bispecific antibody is removed from the blood to allow the final step in which DOTA-Bn complexed with the toxicant kills the tumor cells. Is carried out under sufficient conditions and time.
In some embodiments, the contacting step includes administering a bispecific antibody described herein to the organism, such administration such that unbound bispecific antibody is removed from the blood. To be implemented. In some embodiments, the administering step comprises administering a bispecific antibody described herein in combination with a conjugate comprising DOTA-Bn complexed with a payload, the administration comprising: The payload is delivered to tumor cells. In some embodiments, the administration is performed such that the tumor cells are substantially saturated with the bispecific antibody described herein. In some embodiments, the administration is performed such that the tumor cells are substantially saturated with the bispecific antibody described herein prior to administration of the conjugate. In some embodiments, the administration is performed according to a combination regimen, wherein the combination regimen is at least 10 times better than the therapeutic index observed with a reference regimen where the conjugate is administered as a single step monotherapy. Characterized by a combined therapeutic index.
In some embodiments, administration is by a regimen that includes one or more cycles. In some embodiments, administration is by a regimen comprising, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 cycles or 11 cycles or more.
In some embodiments, the administration is a first administration step in which a bispecific antibody is administered, a second administration step in which the conjugate is administered, and a different complexed with the same conjugate or payload. By a regimen that includes one or more cycles of at least one third administration step in which the DOTA-Bn conjugate is administered. In some embodiments, the first step is performed such that the tumor cells are substantially saturated with the bispecific antibody. In some embodiments, the first administration step is performed before the second administration step and before any third administration step, and further administration of the bispecific antibody is performed within the cycle. Not. In some certain embodiments, the cycle after the first cycle does not involve any bispecific antibody administration. In some embodiments, administration is performed over a period of hours to days.

In some embodiments, the conjugate is administered over several minutes.
In some embodiments, the second antigen binding site is based on monoclonal antibody 2D12.5. In some embodiments, the second antigen binding site is based on humanized 2D12.5.
In some embodiments, the present invention provides a method of treating or diagnosing A33 positive cancer in a subject animal. The method includes administering to a subject animal a bispecific antibody described herein, wherein the administration localizes the bispecific antibody to one or more tumors that express an A33 antigen. Conditions and sufficient time, after which a remover is administered to the subject animal (where the remover removes unbound bispecific antibody) followed by radiolabeled DOTA-Bn Administer to animals. In some certain embodiments, the method further comprises the step of administering the bispecific antibody again to the subject animal.
In some embodiments, the step of re-administering the bispecific antibody to the subject animal is performed after administration of the removal agent. In some embodiments, the step of re-administering the bispecific antibody to the subject animal is followed by re-administration of the remover to the subject animal.
In some embodiments, the treatment methods described herein do not substantially cause radiation damage to normal tissue. In some embodiments, the treatment methods described herein result in an increase in the therapeutic index that is greater than 10-fold. In some embodiments, the treatment methods described herein have a curative effect.
In many embodiments, the removal agent is a dextran-based removal agent. In many embodiments, the radiolabeled DOTA is ¹⁷⁷ Lu-DOTA-Bn, ⁹⁰ Y-DOTA-Bn, or ⁸⁶ Y-DOTA-Bn.
In some embodiments, the payload is a toxic payload. In some embodiments, the payload is a biological response modifier. In some embodiments, the payload is selected from the group consisting of a detectable moiety and an active moiety. In some embodiments, the payload is or comprises a group selected from the group consisting of radioisotopes, peptides, nucleic acids, small molecules, nanoparticles, viruses and combinations thereof.
The drawings contained herein, consisting of the following figures, are for illustration only and are not intended to be limiting.

In vitro evaluation of the biochemical purity of huA33-C825 by SE-HPLC chromatography (UV 280 nm). The main peak at 7.583 minutes is a bispecific antibody with a perfect pair with a molecular weight of about 210 KDa. FIG. 5 shows an exemplary Biacore sensorgram of an antibody that binds to human A33 antigen (A) and BSA- (Y) -DOTA-Bn (B). The following exemplary activities are shown: (A) Activity of lutetium-177 against tumors and various normal tissues in various doses of scavenger (CA) and control (saline). Activity is expressed as percent injected dose per gram of tissue (% / ID / g). (B) Activity of lutetium-177 at various concentrations of CA. Activity is expressed as percent injected dose per gram of tissue (% / ID / g). Tumors have extended residence times of several hours and removal is rapid in other tissues. The following exemplary activities are shown: (C) Tumor to organ ratios at various concentrations of CA. (D) Activity of lutetium-177 against time after injection. Activity is expressed as percent injected dose per gram of tissue (% / ID / g). Tumors have extended residence times of several hours and removal is rapid in other tissues. FIG. 6 shows exemplary tumor uptake of ¹⁷⁷ Lu-DOTA-Bn as a function of radioactivity as a function of radioactivity in the treatment of mice bearing human colon cancer xenografts (SW1222). (A) Uptake of ¹⁷⁷ Lu-DOTA-Bn in various tissues; (B) Uptake of ¹⁷⁷ Lu-DOTA-Bn in SW1222 tumor; saturation is achieved after about 40 MBq of injection activity. FIG. 6 shows exemplary tumor uptake of ¹⁷⁷ Lu-DOTA-Bn as a function of radioactivity as a function of radioactivity in the treatment of mice bearing human colon cancer xenografts (SW1222). (C) Activity of ¹⁷⁷ Lu-DOTA-Bn in SW1222 tumors and kidneys 24 hours after injection per ¹⁷⁷ Lu-DOTA-Bn injection; (D) 24 hours after injection per ¹⁷⁷ Lu-DOTA-Bn injection Combined ¹⁷⁷ Lu-DOTA-Bn picomoles. Saturation is achieved after about 40 MBq of injection activity. 2 shows exemplary tumor growth measurements (mm ³ ) for groups of mice that have received a single cycle of PRIT. (A) Control (no treatment, n = 8); (B) 0.9 mCi (33.3 MBq; 1 mCi = 37 MBq) ¹⁷⁷ Lu-DOTA-Bn only (n = 6); (C) huA33-C825 + 0.3 mCi ¹⁷⁷ Lu- DOTA-Bn (n = 8); (D) huA33-C825 + 0.9 mCi ¹⁷⁷ Lu-DOTA-Bn (n = 8). Control and treatment (huA33-C825) SW1222 tumors show that the growth pattern of SW1222 tumors is minimally affected (even if the dose is increased) at a single dose of radioactivity. The highest dose (0.9 mCi) shows some effect. The arrow indicates the day on which ¹⁷⁷ Lu-DOTA-Bn was administered. The dose of huA33-C825 was administered at t = -24 hours, followed by dextran-removal agent at t = -4 hours, and ¹⁷⁷ Lu-DOTA-Bn at t = 0 hours. 2 shows exemplary tumor growth measurements (mm ³ ) for mice that received two cycles of PRIT. (A) 2xhuA33-C825 PRIT + 11.1MBq (total 22.2MBq); (B) 2xhuA33-C825 PRIT + 33.3MBq (total 66.6MBq); (C) 2xhuA33-C825 PRIT + 55.5MBq (total 111MBq); (D) 1xhuA33-C825 PRIT + 111MBq (total 111MBq). For two-cycle PRIT performed on days 10 and 17 after tumor inoculation, there was a significant response at all dose levels and a complete response was observed in a dose-dependent manner. In FIG. 6C (2 × huA33-C825 PRIT + 55.5 MBq (total 111 MBq)), all tumors responded to treatment. Toxicity was determined by histopathological assessment of liver, kidney, spleen and bone marrow by comparative pathology MSKCC Lab with monitoring of weight and overall appearance at least 3 times a week. There was no detectable toxicity in these mice. FIG. 4 shows an exemplary tumor response curve summary of DOTA-PRIT multicycle therapy with nude mice bearing SW1222 colon cancer tumors. Three treatment limbs are shown: no treatment (triangle); ¹⁷⁷ Lu-DOTA-Bn alone (square); and 3 cycles of DOTA-PRIT treatment with 55 MBq of ¹⁷⁷ Lu-DOTA-Bn (administered 165 MBq total) Activity (circle), each treatment is indicated by an arrow below the x-axis). 2 shows exemplary maximum intensity nanoSPECT / CT images and activity concentrations in the tumor over time. Images were collected from SW1222 tumor-bearing nude mice treated with a single cycle of anti-GPA33 PRIT + 55MBq ¹⁷⁷ Lu-DOTA-Bn and imaged by nanoSPECT / CT at 1, 24 and 160 hours after injection of ¹⁷⁷ Lu-DOTA-Bn It became. Maximum intensity nanoSPECT / CT images of the lower flank region (where the tumor is localized) are shown. The active concentration of the tumor was determined by problem area analysis of the adjusted images.

Definitions The scope of the present invention is defined by the claims appended hereto, and is not limited by the specific embodiments described herein. Those skilled in the art will perceive this disclosure and various modifications that may be equivalent to the described embodiments or that may also be within the scope of the invention.
In general, the terms used herein follow their meaning as understood in the art unless otherwise indicated. Clear definitions of certain terms are provided below, and the meaning of these and other terms in the specific instances herein will be apparent to those skilled in the art from the context.
References cited herein or relevant portions thereof are hereby incorporated by reference.
In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout this specification.
“ Affinity ”: As is known in the art, “affinity” is the amount of tightness that a particular ligand binds to its partner. Affinity can be measured by various methods. In some embodiments, affinity is measured in a quantitative assay. In some such embodiments, the concentration of the binding partner can be fixed to be in excess of the ligand concentration to mimic physiological conditions. Alternatively or additionally, in some embodiments, the binding partner concentration and / or the ligand concentration can be varied. In some such embodiments, affinity can be compared to a reference under comparable conditions (eg, concentration).
As used herein, “ affinity maturation ” (or “ affinity matured antibody ”) refers to an antibody that has one or more modifications in its one or more CDRs, said modifications having no such modifications This results in an improved affinity of the antibody for the antigen compared to the parent antibody. In some embodiments, affinity matured antibodies will have nanomolar affinity, and even even picomolar affinity, for the target antigen. Affinity matured antibodies can be made by any of a variety of procedures known in the art. Marks et al. (Marks et al., 1992, BioTechnology 10: 779-783) describe affinity maturation by _VH and _VL domain shuffling. Random mutagenesis of CDR and / or framework residues is described below: Barbas et al., 1994, Proc. Nat. Acad. Sci. USA 91: 3809-3813; Schier et al., 1995, Gene 169: 147-155; Yelton et al., 1995, J. Immunol. 155: 1994-2004; Jackson et al., 1995, J. Immunol. 154 (7): 3310-9; and Hawkins et al., 1992 , J. Mol. Biol. 226: 889-896.

As used herein, “ amelioration ” refers to the prevention, reduction or alleviation of a condition or the improvement of the condition of a subject animal. Improvement includes, but is not necessarily, complete recovery or complete prevention of a disease, abnormality or symptom (eg, radiation damage).
As used herein, “ animal ” refers to any member of the animal kingdom. In some embodiments, “ animal ” refers to a human of any gender and any stage of development. In some embodiments, “ animal ” refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (eg, rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In certain embodiments, the animal is susceptible to infection by DV. In some embodiments, the animal may be a transgenic animal, a genetically engineered animal, and / or a clone.
As used herein, “ antibody ” has the meaning understood in the art and refers to an immunoglobulin (Ig) that specifically binds to a particular antigen. As is known to those skilled in the art, naturally occurring antibodies are typically composed of four polypeptide chains, two heavy (H) chains and two light (L) chains. Each heavy and light chain is comprised of a variable region (abbreviated herein as HCVR or V _H and LCVR or _VL ) and a constant region. The constant region of the heavy chain includes C _H 1, C _H 2 and C _H 3 domains (and optionally C _H 4 domains in the case of IgM and IgE). The constant region of the light chain is composed of one domain (C _L ). The V _H and V _L regions further include hypervariable regions called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). To do. Each V _H and V _L is composed of 3 CDRs and 4 FRs, which are arranged in the following order from the amino terminus to the carboxy terminus (FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 ). The immunoglobulin molecules can be of any type (eg IgM, IgD, IgG, IgA and IgE), any class (eg IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or any subclass.

Antibody factor : As used herein, the term “antibody factor” refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide having sufficient immunoglobulin structural elements to confer specific binding. In various embodiments, suitable antibody agents include monoclonal antibodies, polyclonal antibodies, humanized antibodies, primatized antibodies, chimeric antibodies, human antibodies, bispecific or multispecific antibodies, single domain antibodies (eg, Shark single domain antibodies (eg IgNAR or fragments thereof), complexed antibodies (ie antibodies conjugated or fused to other proteins, radiolabels, cytotoxins), small modular immunopharmaceuticals ( "SMIP ^™ ")), single chain antibodies, camelid antibodies, antibody fragments, etc. In some embodiments, the term can refer to staple peptides. In embodiments, the terms can refer to antibody-like binding peptidomimetics, hi some embodiments, the terms In some embodiments, the term can refer to monobodies or adnectins, hi many embodiments, antibody factors can be determined by one of skill in the art as complementarity determining regions (CDRs). Or a polypeptide whose amino acid sequence comprises one or more structural elements that are recognized as), or in some embodiments, an antibody agent has at least one CDR whose amino acid sequence is (Eg, at least one heavy chain CDR and / or at least one light chain CDR) (substantially identical to that found in a reference antibody) or comprising such a polypeptide. In such embodiments, the included CDR is substantially identical to the reference CDR, i.e., the CDR is identical in sequence to the reference CDR or from 1 to 5 amino acids. In some embodiments, the included CDR is substantially identical to the reference CDR, ie, the CDR is at least 85%, 86%, 87%, 88%, 89% of the reference CDR. , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, In some embodiments, the included CDRs are Substantially identical to the reference CDR, ie, the CDR exhibits at least 96%, 97%, 98%, 99% or 100% sequence identity with the reference CDR, In some embodiments, the included CDR Is substantially identical to the reference CDR, i.e. at least one amino acid in the included CDR is deleted, added or substituted compared to the reference CDR, otherwise the CDR is It has the same amino acid sequence as that of the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR, i.e., 1 to 5 amino acids in the included CDR are deleted or added or substituted relative to the reference CDR. Otherwise, the included CDR comprises the same amino acid sequence as the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR, i.e., at least one amino acid in the included CDR is substituted as compared to the reference CDR, but otherwise included The CDR has an amino acid sequence identical to the amino acid sequence of the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR, i.e., 1 to 5 amino acids in the included CDR are deleted, added, or substituted relative to the reference CDR. Otherwise, the included CDR has the same amino acid sequence as the reference CDR. In some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those of skill in the art as an immunoglobulin variable domain. In some embodiments, the antibody agent is a polypeptide having a binding domain, wherein the binding domain is homologous or largely homologous to an immunoglobulin binding domain. In some embodiments, the antibody agent is a polypeptide comprising all CDRs found in one or more specific reference antibody chains (eg, heavy and / or light chain), or such poly Contains peptides.

As used herein, an “ antibody component ” refers to a polypeptide element that specifically binds to an epitope or antigen and includes one or more immunoglobulin structural features, said element being a complete polypeptide. But it may also be part of a larger polypeptide (eg, a fusion polypeptide described herein). In general, an antibody component is any polypeptide whose amino acid sequence contains elements characteristic of the binding region of an antibody (eg, the region is a light chain or variable region of an antibody or one or more of the foregoing). Complementarity-determining regions (" CDRs "), or antibody heavy chains or variable regions or one or more of the CDRs (optionally including one or more framework regions)). In some embodiments, the antibody component is or comprises a full length antibody. In some embodiments, the antibody component is less than full length but comprises at least one binding site (the binding site comprising at least one (preferably at least 2) having a structure known as an antibody “ variable region ”. One) array). In some embodiments, the term “ antibody component ” includes any protein having a binding domain, said protein being homologous or largely homologous to an immunoglobulin binding domain. In a specific embodiment, an included “antibody component” includes a polypeptide having a binding domain that exhibits at least 99% identity with an immunoglobulin binding domain. In some embodiments, the included “antibody component” comprises an immunoglobulin binding domain (eg, a reference immunoglobulin binding domain) and at least 70%, 75%, 80%, 85%, 90%, 95% or 98% Any polypeptide having a binding domain that exhibits identity. The included “ antibody component ” can have an amino acid sequence identical to the amino acid sequence of an antibody (part thereof, eg, antigen binding portion thereof) found in a natural source. Antibody components can be monospecific, bispecific or multispecific. The antibody component can include structural elements characteristic of any immunoglobulin class, including any of the human classes (IgG, IgM, IgA, IgD and IgE). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of such antibodies can also be bispecific, bispecific, or multispecific formats that specifically bind to two or more different antigens. Examples of binding fragments encompassed by the term “ antigen-binding portion ” of an antibody include: (i) a monovalent fragment consisting of a Fab fragment, V _H , V _L , C _H 1 and C _L domains; ii) F (ab ′) ₂ fragment, a bivalent fragment comprising two Fab fragments joined by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of V _H and C _H 1 domains; An Fv fragment consisting of one arm _VH and _VL domains; (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546), which contains only one variable domain; and (vi) Isolated complementarity determining region (CDR). Furthermore, the two domains (V _H and V _L ) of the Fv fragment are encoded by separate genes, but they are monovalent molecules (V _H and V _L regions paired together using recombinant methods). Known as single chain Fv (scFv) (see, eg, Bird et al., 1988, Science 242: 423-426; and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879 The two domains can be joined by a synthetic linker that can be a single protein chain forming -5883)). In some embodiments, an “ antibody component ” as described herein is such a single chain antibody or comprises such a single chain antibody. In some embodiments, the “ antibody component ” is or comprises a diabody. Diabodies are bivalent bispecific antibodies, where the V _H and V _L domains are expressed as only one polypeptide chain, but cannot pair between the two domains on the same chain Shorter linkers are used, so these domains must pair with the complementary domains of another chain, resulting in two antigen binding sites (see, eg, Holliger, P., et al. 1993, Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, RJ, 1994, Structure 2 (12): 1121-1123). Such antibody binding moieties are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp., ISBN 3-540-41354-5). In some embodiments, the antibody is a single chain “ linear antibody ” or comprises such an antibody. The linear antibody comprises a pair of vertically aligned Fv segments (V _H -C _H 1 -V _H -C _H 1), which together with a complementary light chain polypeptide It forms the antigen binding region (Zapata et al., 1995, Protein Eng. 8 (10): 1057-1062; and US Pat. No. 5,641,870). In some embodiments, antibody components can have structural elements that have the characteristics of chimeric or humanized antibodies. In general, humanized antibodies are derived from the CDRs of non-human species (eg, mouse, rat or rabbit, donor antibody) whose recipient complementarity determining regions (CDRs) have the desired specificity, affinity and performance. Is a human immunoglobulin (recipient antibody) that is replaced by In some embodiments, the antibody component can have a structural element having the characteristics of a human antibody.

As used herein, “ biological activity ” refers to an observable biological action or outcome achieved by a factor or entity in question. For example, in some embodiments, the specific binding interaction is a biological activity. In some embodiments, the modulation (eg, induction, enhancement or inhibition) of a biological pathway or event is a biological activity. In some embodiments, the presence or degree of biological activity is assessed by detection of direct or indirect products produced by the biological pathway or event in question.
As used herein, a “ bispecific antibody ” refers to a bispecific binding agent, wherein at least one (typically both) of the binding moieties is an antibody component or an antibody Contains components. A variety of different bispecific antibody constructs are known in the art. In some embodiments, each binding portion of a bispecific antibody that is or comprises an antibody component comprises a V _H and / or _VL region, and in some such embodiments, V _H and / or V _L regions are those found in individual monoclonal antibodies. In some embodiments, where the bispecific antibody comprises a binding portion of two antibody components, each antibody component comprises a V _H and / or _VL region derived from a different monoclonal antibody. In some embodiments, where the bispecific antibody comprises a binding portion of two antibody components, one of the binding portions of the two antibody components is a V _H comprising a CDR from the first monoclonal antibody. And / or an immunoglobulin molecule having a _VL region, and one of the binding parts of the two antibody components includes a V _H and / or _VL region containing a CDR derived from the second monoclonal antibody Fragments (eg, F (ab ′), F (ab ′) ₂ , Fd, Fv, dAB, scFv, etc.) are included.
As used herein, “ bispecific binding agent ” refers to a polypeptide having two separate binding moieties, each of which binds to a distinct target. In some embodiments, the bispecific binding agent is only one polypeptide or comprises only one polypeptide, and in some embodiments, the bispecific binding agent is a plurality of peptides Or in some such embodiments, they may be covalently linked to each other, eg, by cross-linking. In some embodiments, the two binding moieties of the bispecific binding agent recognize different sites (eg, epitopes) of the same target (eg, antigen), and in some embodiments they recognize different targets. . In some embodiments, the bispecific binding agent can bind simultaneously with two targets of different structures.

As used herein, “ carrier ” refers to an adjuvant, excipient, or vehicle with which the composition is administered. In some exemplary embodiments, the carrier can include sterile liquids such as water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. included. In some embodiments, the carrier is one or more solid components or includes one or more solid components.
As used herein, “ CDR ” refers to a complementarity determining region within an antibody variable region. There are three CDRs in each of the variable regions of the heavy and light chains, referred to as CDR1, CDR2, and CDR3 for each of the variable regions. A “ CDR set ” or “ CDR set ” refers to a group of three or six CDRs present in only one variable region capable of binding to an antigen or cognate heavy and light chain variable regions capable of binding to an antigen. Certain systems that define CDR boundaries have been established in the art (eg, Kabat, Chothia, etc.). Those skilled in the art will understand the differences between these systems and understand the CDR boundaries to the extent necessary to understand and implement the present invention.
As used herein, a “ CDR grafted antibody ” comprises heavy and light chain variable region sequences whose amino acid sequences are derived from one species, but one or more CDR regions of V _H and / or V _L. An antibody in which the sequence is replaced with a CDR sequence of another species, eg, an antibody having a murine _VH and / or _VL region in which one or more murine CDRs (eg, CDR3) are replaced with human CDR sequences. Similarly, “ CDR-grafted antibody ” can also refer to an antibody having human V _H and V _L regions in which one or more human CDRs (eg, CDR3) have been replaced with mouse CDR sequences.
As used herein, a “ chimeric antibody ” is a _VH and _VL region sequence that is found in a first species and a constant region that is found in a second species (different from the first species). Refers to an antibody comprising a sequence. In many embodiments, the chimeric antibody has murine _VH and _VL regions linked to a human constant region. In some embodiments, an antibody having human _VH and _VL regions linked to a non-human constant region (eg, a mouse constant region) is referred to as a “reverse chimeric antibody”.

“ Combination therapy ”: As used herein, the term “combination therapy” refers to a situation where a subject animal is simultaneously exposed to two or more treatment regimens (eg, two or more therapeutic factors). In some embodiments, such factors can be administered continuously. In some embodiments, such factors are administered in overlapping dosing regimens.
As used herein, “ comparable ” refers to two or more that are not identical to each other but are sufficiently similar to allow comparison and therefore reasonably draw conclusions based on observed differences or similarities Refers to a set of factors, entities, situations, and conditions. To the extent that two or more such factors, entities, situations, condition sets, etc. are considered comparable, one skilled in the art will understand from the context how much identity is required in any given environment. Like.
As used herein, “ corresponding to ” indicates the position / identity of an amino acid residue within the polypeptide of interest. For brevity, residues within a polypeptide are often shown using a standard numbering system based on the polypeptide associated with the reference, and thus, for example, the residue at position 190 in a particular amino acid chain The “ corresponding ” amino acid need not actually be the 190th amino acid, but will be understood by those skilled in the art to correspond to the residue found at the 190th position in the reference polypeptide (the “ corresponding ” amino acid Those skilled in the art will readily understand how to identify them).
As used herein, a “ detection agent ” is a moiety or action that facilitates detection due to, for example, specific structural and / or chemical characteristics of the moiety or agent, and / or their functional properties. Refers to a factor. Non-limiting examples of such factors include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands ( For example, biotin). Many detection agents are known in the art, such as systems for their binding to antibodies (see, eg, US Pat. Nos. 5,021,236, 4,938,948 and 4,472,509, each of which is herein incorporated by reference. include)). Specific examples include paramagnetic ions, radioisotopes, fluorescent dyes, NMR detectable substances, and X-ray imaging agents, among others. In some embodiments of the invention, the complexed detection agent is a diagnostic or imaging agent.

“ Dosage form ” and “ unit dosage form ”: As used herein, the term “dosage form” refers to a physically separated therapeutic agent unit to treat a subject animal (eg, a human patient). Point to. Each unit contains a predetermined amount of active substance calculated or revealed to produce the desired therapeutic effect when administered according to an appropriate dosing regimen for the appropriate population. For example, in some embodiments, such an amount is administered in a dosage regimen that has been determined to correlate with a desired or beneficial outcome when administered to an appropriate population (ie, having a therapeutic dosage regimen). Unit dosage suitable for. It will be appreciated, however, that the total dosage administered to any individual patient will be selected by a medical professional (eg, a physician) within the scope of careful medical judgment.
As used herein, an “ administration regimen ” (or “ treatment regimen ”) is a set of unit doses that are each administered to a subject animal, typically at intervals (typically two or more times). is there. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which can include one or more doses. In some embodiments, the dosing regimen comprises multiple doses, each separated from each other by the same length of time. In some embodiments, the dosing regimen comprises multiple doses, each dose being separated by at least two different time periods. In some embodiments, the therapeutic agent is administered continuously (eg, by infusion) over a predetermined period of time. In some embodiments, the therapeutic agent is administered once a day (QD) or twice a day (BID). In some embodiments, the dosing regimen comprises multiple doses, each of which is separated from each other by the same length of time. In some embodiments, the dosing regimen comprises multiple doses, each dose being separated by at least two different time periods. In some embodiments, all doses in the dosing regimen are the same unit dose amount. In some embodiments, separate doses in the dosing regimen vary in amount. In some embodiments, the dosing regimen comprises a first dose at a first dose followed by one or more additional doses at a second dose that is different from the first dose. In some embodiments, the dosing regimen comprises a first dose at a first dose followed by one or more additional doses at a second dose that is the same as the first dose. In some embodiments, the dosage regimen correlates with the desired or beneficial outcome when administered to the appropriate population (ie, when it is a therapeutic dosage regimen).

As used herein, “ effector function ” refers to a biochemical event resulting from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include, but are not limited to, antibody dependent cell mediated cytotoxicity (ADCC), antibody dependent cell mediated phagocytosis (ADCP), and complement mediated cytotoxicity (CMC). In some embodiments, the effector function is one that operates after antigen binding, one that operates separately from antigen binding, or both.
As used herein, “ effector cells ” refers to cells of the immune system that express one or more Fc receptors and mediate one or more effector functions. In some embodiments, the effector cells include monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, large lymphoid granulocytes, Langerhans cells, natural killer (NK) cells. , T lymphocytes, B lymphocytes (including but not limited to), which may be derived from any organism, including humans, mice, rats, rabbits and monkeys.
As used herein, “ operated ” refers to the characteristic of being manually manipulated. For example, in some embodiments, a polynucleotide is “ engineered ” when two or more polynucleotide sequences that are not linked together in natural order are directly manipulated and linked directly to each other as engineered nucleotides. it can be considered to have been ". In some such specific examples, the engineered polynucleotide can include regulatory sequences. The regulatory sequence is found in nature in operative relationship with the first coding sequence, but is not operatively related to the second coding sequence and is artificially linked, and thus operatively associated with the second coding sequence. There is a relationship. Alternatively or additionally, in some embodiments, the first and second nucleic acid sequences, each encoding a polypeptide element or domain that are not naturally joined together, are single engineered. They can be linked together as polynucleotides. Similarly, in some embodiments, when a cell or organism is manipulated to alter its genetic information (eg, new genetic material that did not previously exist is introduced, or previously existing genetic material is When modified or removed) a cell or organism can be considered “engineered”. As is common practice and will be understood by those skilled in the art, engineered polynucleotides or progeny of cells are typically still “engineered” even if the actual manipulation was performed on a previous entity. It is said. Furthermore, as will be appreciated by those skilled in the art, a variety of methods are available that can accomplish the “operations” described herein. For example, in some embodiments, “manipulation” is through the use of a computer system programmed to perform analysis or comparison, or alternatively to analyze, recommend and / or sort sequences, modifications (eg, nucleic acids). Selection, design, or selection of sequences, polypeptide sequences, cells, tissues and / or organisms. Alternatively or additionally, in some embodiments, “manipulation” includes in vitro chemical synthesis methods and / or recombinant nucleic acid techniques (eg, nucleic acid amplification (eg, by polymerase chain reaction), hybridization, mutagenesis). , Transformation, etc.) and / or the use of any of a variety of managed mating methods. As will be appreciated by those skilled in the art, a variety of such established techniques (eg, recombinant DNA, oligonucleotide synthesis, and techniques for tissue culture and transformation (eg, electroporation, lipofection, etc.)) It is described in various general and detailed references known in the art and cited and discussed throughout this specification (see, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) (included herein by reference for any purpose)).

As used herein, an “ epitope ” includes any moiety that is specifically recognized by an immunoglobulin (eg, antibody or receptor) binding member. In some embodiments, an epitope is comprised of multiple chemical atoms or chemical groups on an antigen. In some embodiments, such chemical atoms or groups are exposed to the surface when the antigen assumes the relevant three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically close to each other in space when the antigen adopts such a conformation. In some embodiments, at least some such chemical atoms and groups are physically separated from each other when the antigen adopts another conformation (eg, when linearized).
As used herein, “ excipient ” refers to a non-therapeutic factor that can be added to a pharmaceutical composition, for example, to provide or promote a desired invariant or stabilizing effect. Suitable pharmaceutical excipients include, for example: starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, Nonfat dry milk, glycerin, propylene glycol, water, ethanol, etc.
As used herein, “ Fc ligand ” refers to a molecule (preferably a polypeptide) derived from any organism that binds to the Fc region of an antibody to form an Fc-ligand complex. Fc ligands include (but are not limited to): FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16A), FcγRIIIB (CD16B), FcγRI (CD64), FcεRII (CD23), FcRn, Clq, C3, Staphylococcus protein A, Streptococcus protein G, and viral FcγR. Fc ligands can include undiscovered molecules that bind to Fc.
“ Fluorescent labels ” known in the art have a fluorescent nature and in some embodiments are moieties or entities that can be detected based on such fluorescence. In some embodiments, the fluorescent label may include or may not include one or more of the following: Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL , BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5, 6-FAM, Fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG , Rhodamine green, rhodamine red, renographin, ROX, TAMRA, TET, tetramethylrhodamine, and / or Texas Red.

As used herein, “ framework ” or “ framework region ” refers to a variable region sequence excluding CDRs. Since CDR sequences can be determined by various systems, similarly, framework sequences are subject to different interpretations accordingly. Six CDRs divide the framework regions on the heavy and light chains into four subregions (FR1, FR2, FR3 and FR4) on each chain. In this case, CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. Without identifying individual subregions as FR1, FR2, FR3, or FR4, as others say, the framework region is a group of FRs grouped within the variable region of a naturally occurring single immunoglobulin chain. Represent. As used herein, FR represents one of four subregions, for example FR1 is the first framework region closest to the amino terminus of the variable region and 5 ′ to CDR1. FR represents two or more sub-regions constituting the framework region.
As used herein, “ host cell ” refers to a cell into which exogenous DNA (recombinant or otherwise) has been introduced. Those skilled in the art reading this disclosure will appreciate that such terms refer not only to the individual subject cells, but also to the progeny of such cells. In fact, such progeny may not be identical to the parental cell, as certain modifications may occur in subsequent generations due to mutations or environmental effects, but the foregoing is still Included within the scope of the term “ host cell ” as used herein. In some embodiments, host cells include prokaryotic and eukaryotic cells selected from any of the life worlds appropriate for expression of exogenous DNA (eg, recombinant nucleic acid sequences). Exemplary cells include: prokaryotic and eukaryotic cells (single or multicellular), bacterial cells (eg, strains of E. coli, Bacillus, Streptomyces, etc.), myco Bacterial cells, fungal cells, yeast cells (eg S. cerevisiae, S. pombe, P. pastoris, P. methanolica), plant cells Insect cells (eg, SF-9, SF-21, baculovirus infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions (eg, hybridoma or hybridoma-hybridoma fusion) cell). In some embodiments, the cell is a human cell, monkey cell, ape cell, hamster cell, rat cell or mouse cell. In some embodiments, the cell is a eukaryotic cell and is selected from the following cells: CHO (eg, CHO Kl, DXB-1 1 CHO, Veggie-CHO), COS (eg, COS-7), retinal cell , Vero, CV1, kidney (eg HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60 (eg BHK21), Jurkat, Daudi, A431 (Epidermis), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Sertoli cells, BRL3A cells, HT1080 cells, myeloma cells, tumor cells, and the aforementioned cells Cell lines. In some embodiments, the cell comprises one or more viral genes (eg, retinal cells that express the viral gene, eg, PER.C6 ^™ cells).

As used herein, “ human antibody ” is intended to include antibodies having variable and constant regions made (or assembled) from human immunoglobulin sequences. In some embodiments, the amino acid sequence of the antibody (or antibody component) includes, for example, one or more CDRs (especially in CDR3) that contain residues or elements that are not encoded by human germline immunoglobulin sequences. (Eg, even if they contain sequence changes originally introduced in vitro by random mutagenesis or site-directed mutagenesis, or in vivo by somatic mutagenesis), they are “ human antibodies ”. Can be considered.
“ Humanized ” as known in the art: The term “ humanized ” includes the V _H and V _L region sequences from which the amino acid sequence of the antibody is derived from a reference antibody generated in a non-human species (eg, mouse), Furthermore, it refers to antibodies intended to contain modifications to their sequences relative to the reference antibody and to make them more “ human-like ” (ie, more similar to human germline variable sequences). . In some embodiments, a “ humanized ” antibody (or antibody component) binds immunospecifically to an antigen of interest and has a substantially amino acid sequence, such as the amino acid sequence of a human antibody ( FR) region and an antibody having a complementarity determining region (CDR) substantially having an amino acid sequence such as the amino acid sequence of a non-human antibody. A humanized antibody comprises substantially all of at least one (typically two) variable domains (Fab, Fab ′, F (ab ′) ₂ , FabC, Fv), in which the CDR regions All or substantially all of that corresponds to that of a non-human immunoglobulin (ie, a donor immunoglobulin), and all or substantially all of the framework region is of a human immunoglobulin consensus sequence. In some embodiments, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin constant region. In some embodiments, a humanized antibody comprises at least the variable domain of a heavy chain with a light chain. The antibody can also comprise the C _H 1, hinge, C _H 2, C _H 3 and optionally C _H 4 regions of the heavy chain constant region. In some embodiments, the humanized antibody comprises only a humanized _VL region. In some embodiments, the humanized antibody comprises only a humanized _VH region. In some embodiments, the humanized antibody comprises humanized _VH and _VL regions.

As used herein, “ improve ”, “ increase ” or “ decrease ” or the grammatical equivalents above are reference measurements (eg, measurements of the same individual prior to the start of treatment described herein). Or a measurement relative to a single control individual (or multiple control individuals) in the absence of the treatment described herein. A “ control individual ” is an individual who suffers from the same disease or form of injury as the treated individual.
As used herein, “in vitro” refers to events that occur in an artificial environment (eg, in a test tube or reaction vessel, cell culture, etc.) rather than in an environment within a multicellular organism.
As used herein, “in vivo” refers to events that occur in multicellular organisms (eg, humans and non-human animals). In the context of cell-based systems, the term can be used to refer to events that occur in living cells (eg, as opposed to in vitro systems).
As used herein, “ isolated ” refers to (1) from at least some of the components that a substance and / or entity (whether natural and / or laboratory environment) was associated with at the time of initial production. Refers to substances and / or entities that have been isolated and / or (2) artificially designed, produced, prepared, and / or manufactured. Isolated material and / or entities are about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70% of the other components with which they were originally associated, About 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than 99% Can be separated. In some embodiments, the isolated factor is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, More than about 97%, about 98%, about 99% or 99% pure. As used herein, a material is “ pure ” if it is substantially free of other components. In some embodiments, as will be appreciated by those skilled in the art, a substance has been combined with certain other ingredients (eg, one or more carriers or excipients, eg, buffers, solvents, water, etc.) Later, the cells can still be considered “isolated” or even “pure”. In such embodiments, the isolation or percent purity of the material can be calculated without such carriers or excipients. By way of example, in some embodiments, a naturally occurring biological polymer (eg, a polypeptide or polynucleotide) is considered “isolated” if: a) its origin or source For example, some or all of the components that naturally accompany the polymer are not associated with the polymer; b) is substantially free of other polypeptides or nucleic acids of the same species as the species that naturally produces the polymer. C) expressed by a cell or expression system that is not a species that naturally produces the polymer, or is otherwise accompanied by components derived from said cell or expression system. Thus, for example, in some embodiments, a polypeptide that is synthesized chemically or in a cell line different from the cell line that naturally produces the polypeptide is referred to as an “isolated” polypeptide. It is regarded. Alternatively or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques includes: a) other components that naturally accompany the polypeptide, and / or initial production. As such, it can be considered an “isolated” polypeptide to some extent separated from other components associated with the polypeptide.

As used herein, “ K _D ” refers to the dissociation constant from a complex of a binding agent (eg, antibody or binding component thereof) with its partner (eg, an epitope to which the antibody or binding member thereof binds).
As used herein, “ k _off ” refers to the dissociation rate constant of dissociation from a complex of a binding agent (eg, an antibody or binding member thereof) with its partner (eg, an epitope to which the antibody or binding member binds). .
As used herein, “ k _on ” refers to the binding rate constant of binding between a binding agent (eg, an antibody or binding member thereof) and its partner (eg, an epitope to which the antibody or binding member binds).
As used herein, a “ linker ” typically refers to a portion of a molecule or entity that connects two or more different regions of interest (eg, individual structural and / or functional domains or portions). In some embodiments, the linker does not significantly add to the function in question (for example, whether a linker is present in association with the domain or part in question depends on the function of the domain or part). Is not substantially changed). In some embodiments, the linker is characterized by lacking a limited-range structure or rigid structure. In some embodiments, the linker is or comprises a polypeptide, particularly when one or more domains or portions in question are composed of a polypeptide. In some specific embodiments, a polypeptide described herein (eg, an engineered polypeptide) has the general structure S1-L-S2, where S1 and S2 are the moiety or domain of interest. is there. In some embodiments, one or both of S1 and S2 can be or can include a binding element (eg, an antibody component) described herein. In some embodiments, the polypeptide linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or over 100 Can be amino acid long. In some embodiments, the polypeptide linker can have an amino acid sequence that includes or includes a sequence described in the following literature (Holliger, P., et al., 1993, Proc. Natl. Acad. Sci. USA 90: 6444-6448; or Poljak, RJ, et al., 1994, Structure 2: 1121-1123). In some embodiments, the polypeptide linker can be GGGGSGGGGSGGGGS (ie [G ₄ S] ₃ ) or GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (ie [G ₄ S] ₆ ) or have an amino acid sequence comprising said sequence.

As used herein, “ multivalent binding agent ” refers to a binding agent capable of binding two or more antigens, which can be present on the same molecule or on different molecules. The multivalent binding agents described herein are, in some embodiments, engineered to have more than two antigen binding sites, and more typically non-naturally occurring proteins. A multivalent binding agent as described herein refers to a binding agent that can bind to two or more related or unrelated targets. A multivalent binding agent can be composed of multiple copies of a single antibody component or multiple copies of a separate antibody component. Such binding agents can bind to two or more antigens and are tetravalent or multivalent binding agents. The multivalent binding agent can additionally include a therapeutic agent (eg, an immunomodulator, toxin or RNase). The multivalent binding agents described herein can bind at least two targets simultaneously in some embodiments, the targets being different structures, such as two different antigens, two different on the same antigen. An epitope, or hapten and / or antigen or epitope. In many embodiments, a multivalent binding agent of the invention is a protein that has been engineered to have the characteristics of a multivalent binding agent described herein. The multivalent binding agents of the present invention can be monospecific (can bind to one antigen) or multispecific (can bind to more than one antigen), and can further comprise two heavy chain polypeptides and two light chains. It can be composed of chain polypeptides. Each binding site is composed of one heavy chain variable domain and light chain variable domain in some embodiments, with a total of 6 CDRs involved in antigen binding per antigen binding site.

As used herein, “ nucleic acid ” refers to any compound and / or substance that, in its broadest sense, is or can be incorporated into an oligonucleotide chain. In some embodiments, the nucleic acid is a compound and / or substance that is incorporated into or can be incorporated into the oligonucleotide chain via a phosphodiester bond. As will be clear from the context, in some embodiments “ nucleic acid ” refers to individual nucleic acid residues (eg, nucleotides and / or nucleosides), and in some embodiments “ nucleic acid ” refers to individual nucleic acid residues. Refers to the oligonucleotide chain comprising. In some embodiments, “ nucleic acid ” is or comprises RNA. In some embodiments, “ nucleic acid ” is or comprises DNA. In some embodiments, the nucleic acid is one or more natural nucleic acid residues, includes or consists of the foregoing. In some embodiments, the nucleic acid is one or more nucleic acid analogs, includes or consists of the foregoing. In some embodiments, nucleic acid analogs differ from nucleic acids in that they do not utilize phosphodiester linkages. For example, in some embodiments, the nucleic acid is one or more “ peptide nucleic acids ”, includes or consists of the foregoing. Peptide nucleic acids are known in the art and have peptide bonds in the backbone instead of phosphodiester bonds, which are considered within the scope of the present invention. Alternatively or additionally, in some embodiments, the nucleic acid has one or more phosphorothioate and / or 5′-N-phosphoramidite linkages instead of phosphodiester linkages. In some embodiments, the nucleic acid is one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine and deoxycytidine). Consists of. In some embodiments, the nucleic acid is one or more of the following nucleoside analogs, or comprises or consists of: 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine , 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl- Cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0 (6) -methylguanine, 2-thiocytidine, methylated base, Intercalating bases and combinations thereof. In some embodiments, the nucleic acid comprises one or more modified sugars (eg, 2′-fluororibose, ribose, 2′-deoxyribose, arabinose and hexose) relative to the sugar of the natural nucleic acid. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product (eg, RNA or protein). In some embodiments, the nucleic acid comprises one or more introns. In some embodiments, the nucleic acid is isolated one or more times from natural sources, enzymatic synthesis by polymerization based on complementary templates (in vitro or in vivo), regeneration in a recombinant cell or system, And prepared by chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or a residue length exceeding the above. In some embodiments, the nucleic acid is single stranded. In some embodiments, the nucleic acid is double stranded. In some embodiments, the nucleic acid has a nucleotide sequence that includes at least one element that encodes or is the complementary strand of a sequence encoding a polypeptide. In some embodiments, the nucleic acid has enzymatic activity.

As used herein, “ operably linked ” refers to a juxtaposition that permits the described components to function in their intended manner. A control sequence “ operably linked ” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. “ Operably linked ” sequences include both expression control sequences adjacent to the gene of interest and expression control sequences that act in trans (or at a distance) to control the gene of interest. As used herein, the term “ expression control sequence ” refers to a polynucleotide sequence required for the expression and processing of a coding sequence to which the sequence is linked. Expression control sequences include: appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; enhance translation efficiency Sequences that enhance protein stability; and, if desired, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism. For example, in prokaryotic cells, such control sequences generally include a promoter, ribosome binding site, and transcription termination sequence, whereas in eukaryotic cells, such control sequences typically include a promoter and a transcription sequence. Contains an end array. The term “ regulatory sequence ” is intended to include components whose presence is essential for expression and processing, and also includes additional components whose presence is beneficial (eg, leader sequences and fusion partner sequences). be able to.

“ Paramagnetic ions ” as known in the art refer to ions having paramagnetic characteristics. In some embodiments, the paramagnetic ions are chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III). ), Samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), erbium (III), lanthanum (III), gold (III) ), Lead (II), and / or bismuth (III).
As used herein, “ payload ” refers to a portion or entity that is delivered to a site of interest (eg, a cell, tissue, tumor or organism) by binding to another entity. In some embodiments, the payload is or includes a detection agent. In some embodiments, the payload material is or includes a therapeutic agent. In some embodiments, the payload material is or includes a catalytic factor. One skilled in the art will appreciate that the payload material may be of any chemical class. For example, in some embodiments, the payload material is or can include: carbohydrates, isotopes, lipids, nucleic acids, metals, nanoparticles (eg, ceramic or polymer nanoparticles), polypeptides, small molecules , Viruses etc. To name a few, in some embodiments, the therapeutic agent payload is or includes a toxin (eg, a toxic peptide, small molecule or isotope (eg, a radioisotope)). In some embodiments, the detection agent payload is a fluorescent substance or factor, a radioactive substance or factor, a factor or substance detectable by binding (eg, tag, hapten, ligand, etc.), a catalytic factor, or the like, or Including the above.
As used herein, “ physiological conditions ” have the meaning understood in the art to refer to conditions under which cells or organisms survive and / or regenerate. In some embodiments, the term refers to external or internal environmental conditions that may exist naturally for an organism or cell line. In some embodiments, the physiological condition is a condition that exists in the body of a human or non-human animal, particularly a condition that exists at and / or within a surgical site. Physiological conditions typically include, for example, a temperature range of 20 ° C to 40 ° C, 1 atmospheric pressure, 6 to 8 pH, 1 to 20 mM glucose concentration, atmospheric oxygen concentration, and gravity present on the earth. Including. In some embodiments, laboratory conditions are manipulated and / or maintained at physiological conditions. In some embodiments, the physiological condition is present in the organism.

As used herein, “ polypeptide ” refers to any polymer chain of amino acids. In some embodiments, the polypeptide has a naturally occurring amino acid sequence. In some embodiments, the polypeptide has a non-naturally occurring amino acid sequence. In some embodiments, the polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through human action. In some embodiments, the polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, the polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, the polypeptide can include D-amino acids, L-amino acids, or both. In some embodiments, the polypeptide can include only D-amino acids. In some embodiments, the polypeptide can include only L-amino acids. In some embodiments, the polypeptide has one or more pendant groups or other modifications (eg, modified or attached to one or more amino acid side chains) at the N-terminus of the polypeptide. , At the C-terminus of the polypeptide, or in any combination of the foregoing. In some embodiments, such pendant groups or modifications can be selected from the group consisting of acetylation, amidation, lipidation, methylation, PEGylation, etc. (including combinations of the foregoing). In some embodiments, the polypeptide may be cyclic and / or may include a cyclic moiety. In some embodiments, the polypeptide is not cyclic and / or does not include any cyclic moiety. In some embodiments, the polypeptide is linear. In some embodiments, the polypeptide can be or can include a staple polypeptide. In some embodiments, the term “polypeptide” may be attached to the name, activity or structure of a reference polypeptide. In such instances, the term is used herein to refer to a polypeptide that shares the relevant activity or structure and thus can be considered a member of the same class or family of polypeptides. For each such class, the present specification provides exemplary polypeptides within the class whose amino acid sequence and / or function is known, as those skilled in the art will be aware of. In some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class. In some embodiments, a member of a polypeptide class or family has significant sequence homology or identity with a reference polypeptide of that class (in some embodiments, all polypeptides within that class). And share common sequence motifs (eg, characteristic sequence elements) and / or share common activities (at comparable levels or within specified ranges in some embodiments). For example, in some embodiments, a member polypeptide exhibits an overall degree of sequence homology or identity with a reference polypeptide, said homology or identity being at least about 30-40%, often About 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or above and / or , Including at least one region exhibiting very high sequence identity (often over 90%, sometimes more than 95%, 96%, 97%, 98% or 99%) (ie said region is a conserved region, In such embodiments, it is a characteristic sequence element or may comprise a sequence element). Such conserved regions usually include at least 3 to 4, often 20 or more amino acids. In some embodiments, the conserved region is at least a stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 or more consecutive amino acids. Is included. In some embodiments, useful polypeptides can comprise or consist of one fragment of the parent polypeptide. In some embodiments, useful polypeptides can comprise or consist of a plurality of fragments, each of the plurality of fragments having a spacing that is different from the organization found in the polypeptide in question. Found in the same parent polypeptide in the same organization (eg, directly linked fragments in the parent may be spatially separated in the polypeptide in question and vice versa, and / or fragments are problematic) The polypeptide of interest may be present in a different order than the parent), and thus the polypeptide in question is a derivative of the parent polypeptide.

“ Prevent ” or “ prevention ” as used herein in connection with the appearance of a disease, abnormality and / or symptom is a reduction in the risk of developing a disease, abnormality and / or symptom, or a disease, abnormality and / or symptom. Refers to delaying the onset of one or more features of symptoms. Prevention can be considered complete when the onset of disease, abnormality and / or symptoms has been prolonged for a predetermined period of time.
“ Radioisotope ”: As used herein, the term “radioisotope” has its art-recognized meaning to refer to an isotope that undergoes radioactive decay. In some embodiments, the radioisotope is actinium-225, astatine-211, bismuth-212, carbon-14, chromium-51, chlorine-36, cobalt-57, cobalt-58, copper-67, europium- 152, Gallium-67, Hydrogen-3, Iodine-123, Iodine-124, Iodine-125, Iodine-131, Indium-111, Iron-59, Lead-212, Lutetium-177, Phosphorus-32, Radium-223, One or more of radium-224, rhenium-186, rhenium-188, selenium-75, sulfur-35, technetium-99m, thorium-227, yttrium-90 and zirconium-89 may be included.
As used herein, a “ recombinant ” is a polypeptide (eg, an antibody or antibody component described herein, or a multiplex) that has been designed, manipulated, prepared, expressed, produced, or isolated by recombinant means. It is intended to refer to a specific binding agent). Such polypeptides include, for example: polypeptides expressed by recombinant expression vectors transfected into host cells, recombinants, polypeptides isolated from combinatorial human polypeptide libraries (Hoogenboom HR, 1997, TIB Tech 15: 62-70; Azzazy H., and Highsmith WE, 2002, Clin. Biochem. 35: 425-445; Gavilondo, JV and Larrick, JW, 2002, BioTechniques 29: 128-145; Hoogenboom H., and Chames , P., 2000, Immunology Today 21: 371-378), antibodies isolated from animals transgenic for human immunoglobulin genes (Taylor, LD et al., 1992, Nucl. Acids Res. 20: 6287-6295 Little M. et al., 2000, Immunology Today 21: 364-370; Kellermann SA. And Green LL, 2002, Current Opinion in Biotechnology 13: 593-597; Murphy, AJ et al., 2014, Proc. Natl Acad. Sci. USA 111 (14): 5153-5158), or splicing selected array elements to another array element A polypeptide prepared, expressed, produced or expressed by any other means including a licing step. In some embodiments, one or more of such selected sequence elements is found in nature. In some embodiments, one or more of such selected sequence elements is computer designed. In some embodiments, one or more such selected sequence elements result from mutagenesis (eg, in vitro or in vivo) of known sequence elements (eg, natural or synthetic origin). For example, in some embodiments, the recombinant antibody polypeptide is composed of sequences found in the germline of the source organism in question (eg, human, mouse, etc.). In some embodiments, the recombinant antibody has an amino acid sequence resulting from mutagenesis (eg, in vitro or in vivo in a transgenic animal), and thus, in the V _H and V _L regions of the recombinant antibody. The amino acid sequence may not occur naturally within the germline in vivo antibody repertoire.

As used herein, “ recovery ” refers to substantially freeing an agent or entity from, for example, isolation (eg, using purification techniques known in the art) from previously associated components. Refers to the process to do. In some embodiments, an agent or entity is recovered from a natural source and / or a source that includes cells.
As used herein, “ reference ” refers to a standard, control, or other suitable reference that performs the comparison described herein. For example, in some embodiments, the reference is a standard or control factor, an animal, an individual, a population, a sample, a sequence, a sequence of steps, a set of conditions, or a value relative to the factor in question Animals, individuals, populations, samples, sequences, sequences of steps, condition sets or values are compared. In some embodiments, the reference is tested and / or measured substantially simultaneously with the test or measurement in question. In some embodiments, the reference is a historical reference, possibly a reference materialized by an important medium. Typically, as will be appreciated by those skilled in the art, the reference is measured or characterized under conditions comparable to those used in the assessment of the problem.
“ Risk ”: As understood from the context of “ risk ” of a disease, disorder and / or condition, includes the likelihood that a particular individual will develop the disease, disorder and / or condition (eg, radiation damage). In some embodiments, risk is expressed as a percentage. In some embodiments, the risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 to 100% It is. In some embodiments, the risk is expressed as a risk relative to the risk associated with the reference sample or group of reference samples. In some embodiments, the reference sample or group of reference samples has a known risk of disease, abnormality, symptom, and / or event (eg, radiation damage). In some embodiments, the reference sample or group of reference samples is from an individual that is comparable to a particular individual. In some embodiments, the relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

“ Specific binding ” as used herein refers to the ability of a binding agent to identify possible partners in the environment in which binding occurs. A binding agent that interacts with one partner when other potential targets are present is referred to as “ specifically binds ” to the target with which the binding agent interacts. In some embodiments, specific binding is assessed by detecting or measuring the degree of binding between the binding agent and its partner. In some embodiments, specific binding is assessed by detecting or measuring the degree of dissociation of the binding agent-partner complex. In some embodiments, specific binding is assessed by detecting or measuring the ability of the binding agent to compete for another interaction between the partner and another entity. In some embodiments, specific binding is assessed by performing such detection or measurement over a range of concentrations.
As used herein, “ subject animal ” means any mammal (including humans). In certain embodiments of the invention, the subject animal is an adult, adolescent or infant. In some embodiments, the terms “ individual ” or “ patient ” are used interchangeably with “ subject animal ” and are intended to be interchangeable. Also contemplated by the present invention is the administration of a pharmaceutical composition and / or the implementation of an in utero treatment method.
“ Substantially ”: As used herein, the term “ substantially ” refers to a qualitative state that indicates close to all or all of the degree or degree of the feature or characteristic in question. It will be appreciated by those skilled in the biology field that biological and chemical phenomena are completed very rarely and / or do not progress to completion or absolute results are not achieved or avoided. Thus, the term “ substantially ” is used herein to capture the potential lack of completeness that is inherent in many biological and chemical phenomena.
“ Substantial sequence homology ” as used herein refers to a comparison between amino acids or nucleic acids. As will be appreciated by those skilled in the art, two sequences are considered “ substantially homologous ” if they contain residues that are homologous at corresponding positions. Homologous residues can be the same residue. Alternatively, homologous residues may be non-identical residues with suitably similar structural and / or functional characteristics. For example, as is well known to those skilled in the art, certain amino acids are typically “ hydrophobic ” or “ hydrophilic ” amino acids and / or as amino acids having “ polar ” or “ nonpolar ” side chains. being classified. Substitution of one amino acid for another of the same type can often be considered a “homology” substitution. Typical amino acid classifications are summarized in Tables 1 and 2.

  As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms. Such algorithms include those available in commercially available computer programs such as BLASTN for nucleic acid sequences and BLASTP, gap BLAST and PSI-BLAST for amino acid sequences. Examples of such programs are described below: Altschul et al., 1990, J. Mol. Biol., 215 (3): 403-410; Altschul et al., 1996, Methods in Enzymology 266: 460 -80; Altschul et al., 1997, Nucleic Acids Res. 25: 3389-3402; Baxevanis et al., 1998, Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley; and Misener et al., (Eds .), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999 (all of which are incorporated herein by reference). In addition to identifying homologous sequences, the above programs typically provide an indication of the degree of homology. In some embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90 of the corresponding residues of the two sequences. %, At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more Two sequences are considered to be substantially homologous if they are homologous in the group. In some embodiments, the corresponding sequence is a complete sequence. In some embodiments, the corresponding series is at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, At least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350 , At least 375, at least 400, at least 425, at least 450, at least 475, at least 500 or more than 500 residues.

“ Substantial identity ” as used herein refers to a comparison between amino acid or nucleic acid sequences. As will be appreciated by those skilled in the art, if two sequences contain identical residues at corresponding positions, they are generally considered to be “ substantially identical ”. As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms. Such algorithms include those available in commercially available computer programs such as BLASTN for nucleic acid sequences and BLASTP, gap BLAST and PSI-BLAST for amino acid sequences. Examples of such programs are described below: Altschul et al., 1990, J. Mol. Biol., 215 (3): 403-410; Altschul et al., 1996, Methods in Enzymology 266: 460 -80; Altschul et al., 1997, Nucleic Acids Res. 25: 3389-3402; Baxevanis et al., 1998, Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley; and Misener et al., (Eds .), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying identical sequences, the above programs typically provide an indication of the degree of identity. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92% of the corresponding residues of the two sequences, Two sequences are substantially identical if 93%, 94%, 95%, 96%, 97%, 98%, 99% or more residues are identical over a corresponding stretch of residues. It is considered to be. In some embodiments, the corresponding sequence is a complete sequence. In some embodiments, the corresponding series is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 , 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more. In the context of CDRs, “ substantial identity ” refers to at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of the reference CDR. Refers to a CDR having an amino acid sequence.

“ Surface plasmon resonance ” as used herein refers to an optical phenomenon that allows real-time analysis of specific binding interactions, which include, for example, changes in protein concentration within a biosensor matrix, such as Biacore. Detect by using the (BIAcore) system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further explanation see: Jonsson, U., et al., 1993, Ann. Biol. Clin. 51: 19-26; Jonsson, U., et al., 1991, Biotechniques 11: 620- 627; Johnsson, B., et al., 1995, J. Mol. Recognit. 8: 125-131; and Johnnson, B., et al., 1991, Anal. Biochem. 198: 268-277.
As used herein, " therapeutically effective amount " means an amount that produces the desired effect for which administration is performed. In some embodiments, the term refers to the disease, disorder and / or condition when administered to a population suffering from or susceptible to the disease, disorder and / or condition according to a therapeutic dosage regimen. Refers to an amount sufficient for treatment. In some embodiments, a therapeutically effective amount is an amount that reduces the occurrence and / or severity of the disease, disorder and / or condition and / or delays its onset. One skilled in the art will recognize that the term “ therapeutically effective amount ” does not actually require successful treatment in an individual. Rather, a therapeutically effective amount may be an amount that provides a specific desired pharmacological response to a significant number of subjects when administered to a patient in need of such treatment. In some embodiments, a therapeutically effective amount refers to one or more specific tissues (eg, tissues affected by the disease, abnormality and / or symptoms) or fluids (eg, blood, saliva, serum, Sweat, tears, urine, etc.). It will be appreciated by those skilled in the art that in some embodiments, a therapeutically effective amount of a particular agent or therapy can be formulated and / or administered in a single dose. In some embodiments, the therapeutically effective amount can be formulated and / or administered in multiple doses, for example as part of an administration regimen.

“ Transformation ” as used herein refers to any process by which exogenous DNA is introduced into a host cell. Transformation can occur under natural or artificial conditions using a variety of methods well known in the art. Transformation can be by any known method of inserting foreign nucleic acid sequences into prokaryotic or eukaryotic host cells. In some embodiments, specific transformation methods are selected according to the host cell to be transformed, which methods can include, but are not limited to, viral infection, electroporation, mating, lipofection. It is not limited. In some embodiments, “ transformed ” cells are stably transformed in that the inserted DNA can replicate as an autonomously replicating plasmid or as part of a host chromosome. In some embodiments, the transformed cell transiently expresses the introduced nucleic acid for a limited period of time.
As used herein, a “ vector ” refers to a nucleic acid molecule that can transport another nucleic acid to which the molecule is linked. One type of vector is a “ plasmid ”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated to the viral genome. Certain vectors have the ability to replicate autonomously in the host cell into which they are introduced (eg, bacterial vectors having replication of bacterial origin and episomal mammalian vectors). Other vectors (non-episomal mammalian vectors) can be integrated into the host cell genome upon introduction into the host cell, and thereby replicated along with the host genome. Furthermore, certain vectors can direct the expression of operably linked genes. Such vectors are referred to herein as “expression vectors”.

Detailed Description of Certain Embodiments The present invention presents successful constructs of multispecific binding agents (eg, bispecific antibodies) that bind to established antigens of colorectal cancer. In particular, the present disclosure relates to bispecific antibodies (human A33 glycoprotein antigen and benzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA-Bn)). Specifically presents the successful targeting induction of radioimmunotherapy in colorectal cancer, provides specific such bispecific antibodies, and displays its surprising utility and / or effectiveness To do.
In particular, the present invention provides the first successful therapeutic use of bispecific antibodies specifically targeting the human A33 antigen, and is also an improvement of preliminary target-guided radioimmunotherapy for the treatment of A33-expressing tumors. Provide a method of treatment. The present invention also provides a “theranostic” (ie therapeutic and diagnostic) agent for simultaneous scintigraphic imaging and radioimmunotherapy of A33 positive cancer, and more particularly its surprising utility and / or Or present effectiveness.
A33 (a glycoprotein antigen on human colorectal cancer with limited expression in normal tissues) is also present on tumor cell surfaces after prolonged antibody binding, as opposed to rapid physiological turnover of normal intestinal epithelium. Retained (therapeutic index relative to tissue retention inherent to intestinal antigen). Radioactive scintigraphy and radioimmunotherapy (RIT) for advanced colorectal cancer (“CRC”) using directly conjugated antibodies (eg ¹³¹ I-huA33) result in sub-optimal tumor doses and therapeutic indices. (Welt et al., 1994, J. Clin. Oncol. 12: 1561-1571). The present invention encompasses the recognition that both of these deficiencies can be overcome using a multi-step pre-target guided RIT (PRIT) approach. The approach has high affinity for benzyl-1,4,7,10-tetraazacyclododecane-N, N ', N'',N'''-tetraacetic acid (DOTA-Bn) -radioactive metal complex A bispecific tetravalent huA33-C825 bispecific antibody construct having sex is first targeted to the tumor. The disclosure specifically addresses the clearance of unbound huA33-C825 bispecific antibody from circulation followed by injection of ¹⁷⁷ Lu-radiolabeled DOTA-Bn hapten to deliver a tumor killing dose of PRIT to A33-positive tumors. Indicates to do. To provide one specific example, this disclosure is a human colorectal tumor model of SW1222, in which mice with established subcutaneous tumors healed with minimal toxicity to normal tissues (including bone marrow and kidney) It can be done.
Without wishing to be bound by theory, the data provided herein are that in some embodiments, a PRIT regimen using two-cycle administration of the huA33-C825 bispecific antibody is a single Note that it had a profound effect on the tumor compared to the same PRIT regimen using cycle dosing. Furthermore, the present disclosure shows, among other things, that such two-cycle administration of the huA33-C825 bispecific antibody described herein resulted in a complete response in approximately 80% of the animals tested. Furthermore, what is shown herein is a treatment with additional cycles, which showed a highly effective response. For example, the 3-cycle dosing regimen had a curative effect in 10/10 mice without producing detectable toxicity in the target organs (bone marrow, spleen and kidney). Accordingly, the present disclosure, in at least some embodiments, uses a bispecific antibody format that effectively targets human A33 glycoprotein antigens while minimizing clinical or histological radiation damage. Includes the development of improved PRIT regimens that achieve enhanced tumor targeting and / or enhanced tumor removal.

Human colorectal cancer The human A33 antigen is a transmembrane glycoprotein (213 amino acid polypeptide) with a molecular weight of 43 kD, expressed in more than 95% of human colon cancer, with normal expression (colon and intestinal epithelium) limited , Very little is discharged into the circulation. Initially a murine monoclonal antibody (A33) followed by a humanized form (huA33) (King et al., 1995, British J. Cancer 72: 1364-1372), a target for diagnostic and therapeutic radiotherapy It has been found to have ideal specificity, affinity and antibody-antigen uptake and internalization properties for use as inducers. In a clinical study of ¹²⁴ I-huA33 imaging in patients with colorectal cancer, the differential clearance between antigen-positive tumors and the intestine allows the authors to do the initial administration with the non-radioactive bispecific A33 antibody type (or “ We concluded that an alternative multi-step approach would be preferred, including pre-target induction ") followed by administration with a radiolabeled hapten (O'Donoghue et al., 2011, J. Nucl. Med. 52: 1878-1885). The high tumor persistence of the radioactive iodine form of A33 (eg ¹²⁵ I-A33) facilitates a focused study of the internalization properties of the A33 antibody-antigen complex, and the anti-A33 antibody stays on the surface for a long time In some embodiments, such antibodies are highly suitable for preliminary target induction approaches, particularly when normal expression in the intestine is turned over before the last ligand step (Ackerman et al. , 2008, Mol. Cancer Ther. 7 (7): 2233-2240). The inherent physiology that the intestinal epithelium detaches over 1 to 3 days while holding the antigen and bound antibody together is critical if the target antigen is expressed in these normal cells (Scott et al. , 2005, Clin. Cancer Res. 11: 4810-4817). As described herein, in PRIT, unbound antibody is removed from the blood using a scavenger (CA) prior to the final cytotoxic ligand step. The natural detachment of normal enterocytes is functionally equivalent to the removal process in the intestine. Preliminary targeted induction radioimmunotherapy (PRIT) directed against a variety of other tumor-associated antigens has been studied for colorectal cancer. These include: CEA (hMN-14-anti-DTPA-indium + ¹³¹ I-di-DTPA-indium hapten, and recently anti-CEACAM5-anti-histamine-succinyl-glycine “TF2” with ¹⁷⁷ Lu -IMP288 hapten added), TAG-72 (CC49 scFv-streptavidin + ⁹⁰ Y-DOTA-biotin), Ep-CAM ( ⁹⁰ Y-DOTA-biotin added to NR-LU-10-SA).

Therapies that use antibodies to induce toxicants into tumors, such as radioimmunotherapy (RIT) with directly conjugated antibodies, have so far been limited in part due to less than optimal tumor dose and therapeutic index (TI) Only successful. Furthermore, due to the bystander toxicity of normal tissue, dose escalation is not feasible and thus such treatments have only a limited antitumor effect. Therefore, the present invention is based on the following recognition: Since human A33 glycoprotein antigen is present in colorectal cancer and has inherent retention properties, it is toxic to TI and any major organ with double log conversion Developed PRIT methodology to achieve complete remission of colonized xenografts that do not cause high affinity for the first antigen binding site that binds A33 and the DOTA-Bn (metal) complex (eg C825) Effectiveness of human A33 on tumor cells using a bispecific antibody (referred to herein as huA33-C825) having a second antigen binding site with specificity for single chain Fv (scFv) Can be targeted. As described herein, the PRIT methodology uses the SW1222 subcutaneous colorectal cancer xenograft model, using huA33-C825, a dextran-based removal agent (dextran-CA) and a ¹⁷⁷ Lu-radiolabeled DOTA-Bn hapten ( ¹⁷⁷ Lu-DOTA-Bn) was improved in vivo by titrating.
As described herein, the bispecific binding agents of the present invention provide dual functionality in diagnostic imaging / dosimetry and therapeutic applications. Target-guided radiation therapy (referred to as radioimmunotherapy (RIT)) can deliver enough radiation to overcome any tumor resistance as long as the TI is good. Conventional radiolabeled IgG drugs (eg, ⁹⁰ Y-zevalin) have a sub-optimal TI of 3.1, which is the boundary of curative therapy (where hematologic toxicity limits dose). The present invention encompasses the recognition that the antibody target induction step is separated from the payload step in the pre-target induction RIT (PRIT). The present disclosure evaluates one potential additional advantage of PRIT over conventional RIT that, in some embodiments, PRIT can facilitate patient care. In some embodiments, the initial infusion of cold antibody (in some embodiments, administration of a removal agent) can be performed in a physician's office (eg, a managing physician's office). In some embodiments, only a radiolabeled DOTA-Bn step (typically performed following one or more (in some embodiments, all) other steps) is performed by a nuclear medicine expert. But once the radioactivity has disappeared from the body, the patient can then be returned to the supervision of the attending physician (within 24 hours). Thus, we show herein that PRIT is highly effective by taking advantage of the inherent pharmacokinetics of large and small ligands. The present invention shows in particular that the use of a fully humanized PRIT system utilizing DOTA-Bn (Bn = benzyl) has a significant curative potential in a mouse xenograft model. When TI is improved> 10 fold, no clinical or histological toxicity is observed. PRIT dosimetry using PET or SPECT as a therapeutic diagnostic drug has resulted in highly reproducible dose estimates. Although radioactive isotopes first proved the principle, PRIT can be applied to any payload (including nanoparticles, peptides, toxins, drugs and viruses) linked to DOTA-Bn. We applied the PRIT method to target induction to the human A33 antigen because of its high mortality rate in the United States. For example, A33 positive tumors are associated with high mortality in the following cancers: colorectal cancer (49700 deaths per year), gastric cancer (10720 deaths per year) and pancreatic cancer (39590 deaths per year). At present, there is no cure that has a curative effect on these metastatic cancers.

As described herein, we have identified a bispecific antibody called huA33-C825 (variable region sequence of humanized antibody A33 (King et al., 1995, Brit. J. Cancer 72 : 1364-1372) and C825, benzyl-1,4,7,10-tetraazacyclododecane-N, N ', N'',N'''-tetraacetic acid (DOTA-Bn) -radioactive metal complex Developed murine scFv antibody (Orcutt el al., 2011, Nucl. Med. Biol. 38: 223-233) with high affinity for, and has established xenografts of subcutaneous SW1222 human colorectal cancer Revealed improved PRIT methodology in mice. We provide the data provided herein, particularly the improved PRIT described herein, is a tumor to normal tissue ratio of 105: 1 (blood) and 18: 1 (renal) at 24 hours post injection (pi). Note that we provide Furthermore, as described herein, at 2-120 hours post-injection, the biodistribution of ¹⁷⁷ Lu-DOTA-Bn, the estimated absorbed dose of tumor, liver, spleen and kidney to PRIT (cGy / MBq ) Were 65.8, 0.9 (therapeutic index (TI): 73), 6.3 (TI: 10), 6.6 (TI: 10) and 5.3 (TI: 12), respectively. Thus, in some embodiments, a PRIT regimen utilizing the huA33-C825 bispecific antibody described herein provides an improved therapeutic index and optimal tumor dose in the treatment of human colorectal cancer xenografts. To do. We also provide the data provided herein in particular for two-cycle PRIT treatment of established tumors (66.6 or 111 MBq ¹⁷⁷ Lu-DOTA-Bn, see Table 7), 9/9 complete response and 2/9 140 Note that it has been shown to have led to more than a relapse-free survival. Furthermore, in the other 7 animals, the period for tumor size to reach 500 mm ³ was 27 ± 26 days for 66.6 MBq and 40 ± 6 days for 111 MBq versus 13 ± 2 days for untreated mice. There was no clinical or histological evidence of radiation-induced damage. Accordingly, the data provided herein describes herein that the bispecific antibodies described herein are therapeutic agents for cancer characterized by improved efficacy and safety profiles. The multi-step PRIT approach demonstrates that it is possible to deliver safe and effective radiation using the β-emitting isotope ¹⁷⁷ Lu for removal of established colorectal tumors.

Humanized antibodies In some embodiments, the antibodies used in accordance with the present invention are monoclonal antibodies and / or in some embodiments humanized forms of syngeneic anti-A33 antibodies prepared in other species. possible. In some embodiments, the humanized antibody comprises a non-synthetic non-several amino acid in a light or heavy chain (eg, constant region and variable domain framework region) that is not required for antigen binding of a human immunoglobulin. It is an antibody used in place of the corresponding amino acid of the light chain or heavy chain of a human antibody. By way of example, a humanized version of a murine antibody against an antigen contains, in both its heavy and light chains, (1) the constant region of a human antibody; Has CDRs from murine antibodies. In some embodiments, one or more residues of the human framework regions are changed to residues at corresponding positions in the murine antibody, thus conserving the binding affinity of the humanized antibody for that antibody. Such changes are sometimes referred to as “backmutations”. Similarly, positive mutations can be made to revert to murine sequences for the desired reason (eg, stability or binding affinity). Humanized antibodies are generally less likely to elicit an immune response compared to chimeric human antibodies because of their significantly less non-human elements.
In some embodiments, humanized antibodies are produced by recombinant DNA technology. Alternatively or additionally, suitable methods for producing humanized antibodies of the invention are described, for example, as follows: EP0239400; Jones et al., 1986, Nature 321: 522-525; Riechmann et al. , 1988, Nature 332: 323-327; Verhoeyen et al., 1988, Science 239: 1534-1536; Queen et al., 1989, Proc. Nat. Acad. Sci. USA 86: 10029; US Patent 6,180,370 And Orlandi et al., 1989, Proc. Natl. Acad. Sci. In general, transplantation of murine (or other non-human) CDRs into human antibodies is accomplished as follows. CDNA encoding the heavy and light chain variable domains is isolated from the hybridoma. The DNA sequence of the variable domain (including CDR) is determined by sequencing. DNA encoding CDR, inserting the corresponding region of the heavy or light chain variable domain coding sequence of a human antibody, a human (the κ for γ1 and C _L are for example C _H) desired isotype The gene is synthesized by binding to the constant region gene fragment. Humanized heavy and light chain genes are co-expressed in mammalian host cells (eg, CHO or NSO cells) to produce soluble humanized antibodies. In order to facilitate large-scale production of antibodies, it is often desirable to select high expressers using the DHFR gene or GS gene in the production strain. These production cell lines are cultured in a bioreactor or hollow fiber culture system or WAVE technology to produce a bulk culture of soluble antibodies, or transgenic mammals that express antibodies in milk (eg goats, dairy cows or sheep) (See, eg, US Pat. No. 5,827,690).

As described herein, multispecific binding agents (eg, bispecific antibodies) utilize sequences and / or components found in the humanized antibody A33 described by King et al. (1995, supra). Was operated. Other murine A33 antibodies may be humanized (eg, as described herein) or may be utilized in the manipulation of multispecific binding agents described herein. For example, a murine human chimeric expression vector is constructed using cDNA encoding the light and / or heavy chain variable regions of one or more (typically only one) candidate antibody murine anti-A33 antibodies ( In the chimera, the murine A33 antibody variable region is linked to the human IgG1 (for heavy chain) and human kappa (for light chain) constant regions as previously described). Alternatively or in addition to the foregoing, in some embodiments, a novel type of humanized anti-A33 antibody having variant glycosylation, for example, enhances Fc receptor binding (if so desired). And can be made to enhance antigen affinity.
In some embodiments, human acceptor framework domains can be screened by homology matching with human germline sequences to produce humanized anti-A33 antibodies. Using such a screened human acceptor framework, light and heavy chain variable domains can be designed to create and express multiple variants / variants of each.
Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art, including phage display methods using antibody libraries derived from the human immunoglobulin sequences described above. See, for example: US Pat. Nos. 4,444,887 and 4,716,111; and International Patent Application Publications WO 98/46645, WO 98/60433, WO 98/24893, WO 98/16664, WO 96/34096, WO 96/33735. And WO 91/1074, each of which is incorporated herein by reference in its entirety. For preparation of human monoclonal antibodies, the techniques of Cole et al. (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, ed. RA Reisfeld & S. Sell, pp. 77-96, New York, Alan R. Liss) and The technique of Boerder et al. (Boerder et al., 1991, J. Immunol, 147 (l): 86-95) is also available.

Human antibodies produced using other techniques (though retaining the variable regions of the anti-A33 antibodies of the invention) are included in the invention. Alternatively or additionally, human antibodies can also be produced using transgenic mice. Transgenic mice cannot express functional endogenous mouse immunoglobulins, but can express human immunoglobulin genes (see, eg, Lonberg and Huszar, 1995, Int. Rev. Immunol. 13: 65-93; Taylor, LD, et al., 1992, Nucl. Acids Res. 20: 6287-6295; Kellermann SA., And Green LL, 2002, Current Opinion in Biotechnology 13: 593-597; Little M. et al., 2000, Immunol. Today 21: 364-370; Murphy, AJ et al., 2014, Proc. Natl. Acad. Sci. USA 111 (14): 5153-5158). For a detailed discussion of this technique for the production of human and human monoclonal antibodies and protocols for producing such antibodies, see, for example: International Patent Application Publications WO 98/24893, WO 92 / 01047, WO 96/34096, WO 96/33735; European Patent 0 598 877; U.S. Patents 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, 5,886,793, 5,916,771, and 5,939,598 No. 8,502,018, which is hereby incorporated by reference in its entirety.
Furthermore, human monoclonal antibodies can be produced by immunizing mice transplanted with leukocytes, spleen cells or bone marrow cells of human peripheral blood (eg, XTL trioma technology). Fully human antibodies that recognize selected epitopes can be generated using a technique called “guided selection”. In this approach, sorted non-human monoclonal antibodies (eg, murine antibodies) are used as a guide for the selection of fully human antibodies that recognize the same epitope (Jespers et al., 1988, Biotechnol. 12: 899-903).

As used herein, “ anti-A33 antibody ”, “ anti-A33 antibody portion ” or “ anti-A33 antibody fragment ” and / or “ anti-A33 antibody variant ” and the like, in some embodiments, bind A33. A polypeptide-containing entity comprising at least a portion of an immunoglobulin, and in particular, at least one complementarity determination of heavy or light chains found in any of the individual monoclonal antibodies described herein that bind A33. An entity comprising a polypeptide of a region (CDR) or a ligand binding region thereof (typically comprising the entire CDR or portion thereof found in the corresponding chain). In some embodiments, the term includes not only such CDRs, but also other sequences (heavy or light chain variable regions) that can be incorporated into antibodies of the invention of non-murine origin (preferably human origin). Heavy chain or light chain constant region, framework, or sequences found in any of the foregoing). In some specific embodiments, the term “ anti-A33 antibody ” will be clear from the context, but collectively or individually huA33, hA33, A33, humanized antibody A33, humanized A33 and the above Used to refer to a combination and / or relevant fragment or component, domain or region thereof, eg, a single chain variable fragment (eg, huA33 scFv, hA33 scFv, A33 scFv and combinations thereof).
In some embodiments, the humanized antibody modulates, reduces, counters, mitigates, reduces, blocks, inhibits at least one cellular function in vitro, in situ and / or in vitro. Can be arrested and / or prevented (in which case the cells express human A33). As a non-limiting example, a suitable anti-A33 antibody, specific portion or variant binds with high affinity to certain epitopes (especially peptide epitopes of human A33).

Antibody fragments can be generated by enzymatic cleavage, synthesis or recombinant techniques known in the art and / or described herein. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop codon. For example, a combinatorial gene encoding the F (ab ′) ₂ heavy chain portion can be designed to include a DNA sequence encoding the C _H 1 domain and / or hinge region of the heavy chain. The various portions of the antibody can be joined together chemically by conventional techniques, or can be prepared as a stretch of protein using genetic engineering techniques.
In some embodiments, the chimeric or humanized antibody used in accordance with the invention is found in one or more of the plurality of anti-A33 antibodies described herein, and further comprises at least a portion of the antibody or the remainder. Includes antibodies found in one or more human antibodies or derived from human antibodies. Thus, for example, in some embodiments, the human portion of the antibody comprises a framework, C _L , C _H domain (eg, C _H 1, C _H 2, C _H 3), hinge, V _H , V _L region ( Said can be substantially non-immunogenic in humans). In some embodiments, the “human portion” of an antibody utilized in accordance with the description herein may not show 100% identity with the corresponding sequence found in a human antibody of the appropriate source. Will be understood by those of skill in the art upon reading the present disclosure. In some embodiments, as many human amino acid residues as found in the source human antibody are retained as possible so that immunogenicity is negligible, but in various embodiments, humanized residues are required As appropriate or otherwise desired, it can be modified to maximize the humanization of the antibody while simultaneously supporting the antigen binding site formed by the CDRs. In some embodiments, such alterations or variations maintain or reduce immunogenicity in humans or other species compared to unmodified antibodies.
The antibody agents provided herein, including those that are humanized antibodies described herein and / or that utilize humanized antibody sequence elements, are functionally rearranged human immunoglobulins ( Those skilled in the art reading the present disclosure will appreciate that they can be produced by non-human animal cells or prokaryotic or eukaryotic cells capable of expressing (eg heavy and / or light chain) genes. Furthermore, when the antibody agent is a single chain antibody, the antibody agent can include a linker peptide that is not found in a native human antibody. For example, Fv can comprise a linker peptide, which is, for example, 2 to about 20 glycine or other amino acid residues, preferably 8-15 glycine or other amino acid residues, The variable region of the chain is linked to the variable region of the light chain. Such linker peptides are considered to be of human origin.

Antibody humanization can be performed, for example, by synthesis of a combinatorial library containing six CDRs of a non-human target monoclonal antibody fused in-frame with a pool of individual human frameworks. A human framework library containing genes representing all known heavy and light chain human germline genes can be utilized. The resulting combinatorial library can be screened for binding to the antigen of interest. Such an approach may allow screening and / or selection of particularly preferred combinations of fully human frameworks (eg, in the sense of maintaining binding activity to the parent antibody). Subsequently, the humanized antibody can be further optimized by a variety of techniques.
Antibody humanization can be used to evolve mouse or other non-human antibodies into “ fully human ” antibodies. The resulting antibody contains only human sequences and no mouse or non-human antibody sequences while maintaining similar binding affinity and specificity as the starting antibody.
In some embodiments, the anti-A33 human antibody used in accordance with the present invention comprises a variant Fc region, wherein the variant Fc region is at least one amino acid relative to the wild type Fc region (or parent Fc region). Including modifications, so that the molecule has an altered affinity for an Fc receptor (eg, FcγR), provided that the variant Fc region interacts with an Fc-Fc receptor, as disclosed, for example, by Sondermann et al. Based on crystallographic and structural analysis of action, it is conditioned on the absence of substitution at positions that produce direct contact with the Fc receptor (Sondermann et al., 2000, Nature, 406: 267-273 (supra The literature is hereby incorporated in its entirety by reference)). Examples of positions within the Fc region that cause direct contact with an Fc receptor (eg FcγR) are amino acids 234-239 (hinge region), amino acids 265-269 (B / C loop), amino acids 297-299 (C ′ / E loop), and amino acids 327-332 (F / G loop). In some embodiments, an anti-A33 antibody of the invention comprising a variant Fc region comprises a modification of at least one residue that makes direct contact with FcγR based on structural and crystallographic analysis.

In some embodiments, the anti-A33 antibody used in accordance with the present invention is a humanized A33 antibody with altered affinity for activating and / or inhibitory receptors, wherein the modified antibody is Having a variant Fc region with one or more amino acid modifications, wherein the one or more amino acid modifications are substitutions by alanine at position 297, and in some embodiments, substitutions at 239D, 330L, 332E are Enhancing FcR affinity, in some embodiments, substitution of 322K reduces or eliminates FcR binding. In some embodiments, an anti-A33 antibody used in accordance with the present invention has an Fc region with variant glycosylation compared to the parent Fc region. In some embodiments, the variant glycosylation comprises a lack of fucose, and in some embodiments, the variant glycosylation occurs upon expression in GnT1-deficient CHO cells. In some embodiments, the present invention provides bispecific binding agents having a humanized A33 antibody component comprising a variant Fc region characterized by a K322A substitution. In some embodiments, a provided bispecific binding agent comprises an antibody component that exhibits variant glycosylation (eg, unglycosylated) relative to a parent antibody from which the element can be derived. Including. In some such embodiments, such a variant is or comprises a variant Fc region characterized by a K322A substitution. In some embodiments, such variant elements (eg, variant Fc regions) provide for complement activation and complete elimination of FcR binding, which are otherwise pre-targeted guided radioimmunotherapy as described herein. Can damage the tumor cell membrane before the removal agent is added.
In some embodiments, the invention provides an antibody or antibody factor comprising a variant Fc region (ie, the Fc region includes one or more additions, deletions and / or substitutions relative to a suitable reference Fc) And / or use. The Fc region is characterized in that its effector function is altered and / or its affinity for FcR is enhanced or decreased compared to the reference Fc. These modifications are within the skill of the artisan.

Thus, among other things, the present invention provides multispecific binding factors (eg, antibody factors) that include a variant Fc region that binds to one or more FcγRs with greater affinity. Such factors preferably mediate effector function more effectively, as discussed below. In some embodiments, the invention provides multispecific binding agents (eg, antibody agents) that include a variant Fc region that binds with weak affinity to one or more FcγRs. Reduction or elimination of effector function is desirable in certain cases, for example in the case of antibodies where the mechanism of action of the antibody involves blocking or offsetting the target antigen-carrying cell but does not kill the cell. Furthermore, elimination of effector function is desirable in some embodiments when making bispecific antibodies as discussed below. Reduction or elimination of effector function would be desirable in the case of an autoimmune disease (in this case, the FcγR activating receptor would be blocked in effector cells (this type of function would be present in the host cell)). In general, enhanced effector function can be directed to tumors and foreign cells. In some embodiments, effector function can be moved away from tumor cells.
The Fc variants used in accordance with the present invention can be combined with other Fc modifications, including but not limited to effector function alterations. The invention encompasses combining the Fc variants described herein with other Fc modifications to provide cumulative, synergistic or novel properties to the antibody or Fc fusion. In some such embodiments, an Fc variant can enhance the phenotype of a modification combined with that variant. For example, when a Fc variant is combined with a variant known to bind to FcγRIIIA with a higher affinity than a similar molecule containing the wild-type Fc region, the combination with that variant is a much higher multiple of FcγRIIIA affinity. Bring about strengthening.

In accordance with the present invention, in some embodiments, the Fc variants described herein are incorporated into an antibody or Fc fusion and have one or more Fc glycoforms for one molecule comprising the Fc region. To produce an engineered factor (ie, one or more Fc polypeptides to which one or more carbohydrates are covalently added), where the carbohydrate composition of the glycotype is the glycotype of the parent molecule containing the Fc region Is different).
In some embodiments, a multispecific binding agent (eg, an antibody) described herein can include an Fc variant that exhibits variant glycosylation and / or an appropriate reference Fc region (eg, wild type). Can be expressed in glycosylation-deficient cell lines (eg GnT1-deficient CHO cells) so that the Fc region of the factor lacking glycosylation is generated, or in cell lines that are not deficient in glycosylation An Fc region can be expressed.
In some embodiments, the antibody utilized in accordance with the present invention is preferably a suitable reference that binds to the antigen of interest (eg, A33) without altering the functionality of the antibody (eg, binding activity to the antigen). It can have modified glycosylation sites as compared to antibodies. As used herein, a “ glycosylation site ” in an antibody to which an oligosaccharide (ie, a carbohydrate comprising two or more monosaccharides linked together) is specifically and covalently added. Any specified amino acid sequence is included. The oligosaccharide side chains are typically linked to the backbone of the antibody by N- or O-linkages. N-linked glycosylation refers to the addition of an oligosaccharide moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the addition of an oligosaccharide moiety to a hydroxy amino acid (eg, serine, threonine). For example, certain oligosaccharides (including fucose) and Fc-glycotypes lacking terminal N-acetylglucosamine (huA33-IgG1n) can be produced in special CHO cells and show enhanced ADCC effector function.

In some embodiments, the invention encompasses methods of modifying the carbohydrate content of the antibodies of the invention by the addition or deletion of glycosylation sites. Methods for modifying the carbohydrate content of an antibody are well known in the art and are included in the present invention. See, for example: US Patent No. 6,218,149; EP0359096B1; US Patent Publication 2002/0028486; International Patent Application Publication WO 03/035835; US Patent Publication 2003/0115614; US Patent 6,218,149; US Patent 6,472,511 (supra) All of which are hereby incorporated by reference). In some embodiments, the invention includes a method of modifying the carbohydrate content of an antibody of the invention by deletion of one or more endogenous carbohydrate moieties of the antibody. In some embodiments, the invention includes a method of deleting a glycosylation site in the Fc region of an antibody by correcting position 297 from asparagine to alanine.
Engineered glycoforms can be useful for a variety of purposes, including but not limited to enhancing or reducing effector function. Engineered glycoforms can be generated by any method known to those of skill in the art, for example, by using engineered or variant expression strains, for the collaboration of one or more enzymes (eg, DIN-acetylglucosamine transferase III (GnTIII)). Expression can be produced by expressing molecules containing the Fc region in various organisms or cell lines derived from various organisms, or by modifying the carbohydrate after expressing the molecule containing the Fc region. Methods for producing engineered glycoforms are known in the art, including but not limited to those described in the following literature: Umana et al., 1999, Nat. Biotechnol. 17: 176-180; Davies et al., 2001, Biotechnol. Bioeng. 74: 288-294; Shields et al., 2002, J. Biol. Chem. 277: 26733-26740; Shinkawa et al., 2003, J Biol. Chem. 278: 3466-3473; US Patent 6,602,684; US Patent Application 10 / 277,370; US Patent Application 10 / 113,929; International Patent Application Publication WO 00 / 61739A1; WO 01 / 292246A1; WO 02 / 311140A1; WO 02 / 30954A1; POTILLEGENT ^™ technology (Biowa, Inc. Princeton, NJ); GLYCOMAB ^™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland), each of which is incorporated herein by reference in its entirety. . See for example: International Patent Application Publication WO 00/061739; EA01229125 US Patent Application Publication 2003/0115614; Okazaki et al., 2004, JMB, 336: 1239-49 (each of which is incorporated by reference in its entirety) Is included herein).

Multivalent binding agents As will be appreciated by those skilled in the art, a multivalent binding agent is a molecular entity or complex that contains a binding member that specifically binds to two or more targets (eg, epitopes). . A variety of uses have been found in the art for such multivalent binding agents, including therapeutic uses. As will be appreciated by those skilled in the art, tumor cell killing is facilitated by manipulating multivalent binding factors to direct cytotoxic T cells to tumor cells, for example. Examples of tumor antigens include (but are not limited to): alphafetoprotein (AFP), CA15-3, CA27-29, CA19-9, CA-125, calretinin, carcinoembryonic antigen, CD34, CD99 , CD117, chromogranin, cytokeratin, desmin, epithelial protein (EMA), factor VIII, CD31, FL1, glial fibrillary acidic protein (GFAP), macroscopic cystic fluid protein (GCDFP-15), HMB-45 , Human chorionic gonadotropin (hCG), inhibin, keratin, CD45, lymphocyte marker, MART-1 (Melan-A), Myo Dl, muscle specific actin (MSA), neurofilament, neuron specific enolase (NSE), Placental alkaline phosphatase (PLAP), prostate specific antigen, S100 protein, smooth muscle actin (SMA), synaptophysin, thyroid transcription factor-1, thyroglobulin, tumor M2-PK and bi Mentin.
In some embodiments, the multivalent binding agent used in accordance with the present invention is a bispecific binding agent. In many embodiments, such bispecific binding agents can bind to tumor cells. In many embodiments, such bispecific binding agents can bind to human colorectal cancer cells via the A33 antigen expressed on the tumor cell surface.
In some embodiments, a multivalent binding agent (eg, bispecific binding agent) provided by the present invention is an antibody component or comprises an antibody component. A variety of techniques are known in the art for designing, constructing and / or manufacturing multispecific binding agents comprising antibody components.

For example, multivalent binding agents that utilize complete immunoglobulin frameworks (eg, IgG), single chain variable fragments (scFv) or combinations of the foregoing have already been constructed. A bispecific binding factor composed of two scFv units arranged in tandem has been shown to be a clinically successful bispecific antibody modality. In the case of anti-tumor immunotherapy, a bispecific binding factor containing two single-chain variable fragments aligned in tandem links an scFv that binds a tumor antigen with an scFv that binds T3 by binding to CD3 Designed to be. In this way, it was expected that T cells would be recruited to the tumor site and that they could mediate the killing of the tumor cells that make up the tumor by the cytotoxic properties of certain T cells. Examples of such bispecific binding agents targeting CD19 and CD3 of lymphoma have already been performed and are referred to as bispecific T cell binding (or BiTE) (see, eg, below: Dreier et al., 2003, J. Immunol. 170: 4397-4402; Bargou et al., 2008, Science 321: 974-977), animal xenograft studies have already been successful in preventing tumor growth. In human studies, this bispecific binding agent showed an objective tumor response including 5 partial remissions and 2 complete remissions.
Exemplary bispecific binding agents include a first antibody component specific for a tumor antigen and a small molecule hapten (eg, DTPA, IMP288, DOTA, DOTA-Bn, DOTA-desferrioxamine, biotin, fluorescein Or a second antibody specific to that disclosed by Goodwin et al. (Goodwin, DA et al., 1994, Cancer Res. 54 (22): 5937-5946, which is incorporated herein by reference). Those having components are included. Bispecific binding agents can be made, for example, by combining heavy and / or light chains that recognize different epitopes of the same or different antigens. In some embodiments, due to molecular function, the bispecific binding agent binds to one antigen (epitope) in one of its two binding arms (one V _H / V _L pair), and further It binds to different antigens (or epitopes) in its second arm (different V _H / V _L pairs). According to this definition, a bispecific binding agent has two distinct antigen binding arms (both specific and CDR sequences), and is monovalent for each antigen it binds.

In some embodiments, the bispecific binding agents of the invention are characterized by the ability to bind simultaneously to two targets having different structures. In some embodiments, the bispecific binding agent of the invention comprises at least one component that specifically binds to, for example, a B cell, T cell, bone marrow cell, plasma cell, or mast cell antigen or epitope, and It has at least one other component that specifically binds to the target-inducing complex that holds the therapeutic or diagnostic agent.
Bispecific binding agents of the present invention (eg, bispecific antibodies) have certain modalities when used in a multi-step pretargeted-guided radioimmunotherapy (PRIT) method that targets the human A33 antigen. Based on specific insights that may be more beneficial for certain targets (eg tumor antigens). For example, the bispecific antibodies provided herein utilize a complete IgG and scFv combination. Such bispecific antibodies exhibit bivalent binding via IgG elements (eg anti-A33) and bivalent binding via scFv elements (eg anti-DOTA-Bn). As described herein, bispecific antibodies having this format first bind to A33 positive tumor cells via an IgG element (eg, anti-A33), and excess antibody is removed by a scavenger (CA, eg, dextran). Is removed from the blood via a system remover Said step is followed by a step comprising the use of a radiolabeled small molecule hapten (eg ¹⁷⁷ Lu-DOTA-Bn). Exemplary radiolabeled small molecules include ¹²⁴ I and ¹³¹ I in addition to the radioactive lanthanum series such as yttrium and lutetium (eg ⁸⁶ Y, ⁹⁰ Y and ¹⁷⁷ Lu). Furthermore, the bispecific antibodies of the present invention provide both diagnostic and therapeutic target induction features.
In various embodiments, the bispecific binding agent (eg, bispecific antibody) of the present invention is comprised of a first binding member and a second binding member. In many embodiments, the first and second binding members of the bispecific binding agents described herein are each composed of antibody components characterized by different specificities. In many embodiments, the antibody component is selected from Table 8.

In various embodiments, the bispecific binding agent of the invention comprises a first binding member, a second binding member. In various embodiments, the bispecific binding agent of the invention comprises a first binding member, a second binding member, and a linker that connects both the first and second binding members (eg, the first binding member). And a second coupling element).
In various embodiments, the first and / or second binding member described herein is an antibody component or comprises an antibody component. In various embodiments, the first and / or second binding elements described herein include a linker sequence.
In some embodiments, the linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, Or an amino acid exceeding the above. In some embodiments, the linker tends to not adopt a rigid three-dimensional structure, but rather is characterized by providing flexibility to the peptide (eg, the first and second binding elements). In some embodiments, the linker is based on a special property imparted to the bispecific binding agent (eg, reduced aggregation and / or enhanced stability), as described herein. Used in In some embodiments, the bispecific binding agents of the invention comprise a G ₄ S linker. In some embodiments, the bispecific binding agent of the invention comprises a (G ₄ S) _n linker, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 or 16 or more).
In various embodiments, the first and / or second binding member described herein is or includes an immunoglobulin (eg, IgG). In various embodiments, the first and / or second binding member described herein comprises an antibody fragment (eg, scFv) or is an antibody fragment. In various embodiments, the first binding member described herein comprises an immunoglobulin and the second binding member comprises an antibody fragment. In some certain embodiments, the first binding member is an immunoglobulin and the second binding member is an antibody fragment. In some certain embodiments, the first binding member is IgG and the second binding member is scFv.

In some certain embodiments, the bispecific binding agents of the invention comprise immunoglobulins, which immunoglobulins include heavy and light chains and scFV. In some certain embodiments, the scFv is linked to the C-terminus of the immunoglobulin heavy chain. In some certain embodiments, the scFv is linked to the C-terminus of the immunoglobulin light chain. In various embodiments, the scFv is linked to the heavy or light chain via a linker.
In some embodiments, a bispecific binding agent of the invention has at least about 50% (eg, at least about 55%, 60%, 65%, 70%, 75) with one or more sequences listed in Table 8. %, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) containing one or more sequences that are identical.
In some embodiments, a bispecific binding agent of the invention comprises one or more sequences that are substantially identical to one or more sequences listed in Table 8.
In some embodiments, the bispecific binding agents of the invention comprise one or more sequences that are identical to one or more sequences listed in Table 8.
In some embodiments, the bispecific binding agents of the invention are selected from one or more sequences listed in Table 8. In some certain embodiments, the bispecific binding agents of the invention are selected from the two sequences set forth in Table 8, such as heavy and light chain sequences.
In various embodiments, the first binding member of the bispecific binding agent described herein is at least about 50% (eg, 50%, 55%, 60%, etc.) with the antibody component listed in Table 8. 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) Antibody components having sequences that are identical are included.
In various embodiments, the first binding member of the bispecific binding agent described herein comprises an antibody component having a sequence that is substantially identical to an antibody component listed in Table 8.
In various embodiments, the first binding member of the bispecific binding agent described herein comprises an antibody component having the same sequence as the antibody components listed in Table 8.
In various embodiments, the second binding member of the bispecific binding agent described herein is at least about 50% (eg, 50%, 55%, 60%, etc.) with the antibody component listed in Table 8. 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) Antibody components having sequences that are identical are included.
In various embodiments, the second binding member of the bispecific binding agent described herein comprises an antibody component having a sequence that is substantially identical to the antibody component described in Table 8.
In various embodiments, the second binding member of the bispecific binding agent described herein comprises an antibody component having the same sequence as the antibody components listed in Table 8.

As described herein, we have developed an improved multi-step PRIT using a bispecific antibody designated huA33-C825 in immunodeficient mice bearing A33 positive human colorectal tumor (SW1222). Provide law. Such methods include simultaneous injection of various doses of huA33-C825 and dextran-based CA followed by injection of ¹⁷⁷ Lu-DOTA-Bn therapeutic diagnostic (ie diagnostic and therapeutic) hapten with simultaneous scintigraphic imaging. Include for chemical and radioimmunotherapy. The present invention specifically relates to tumor uptake as a function of biodistribution studies that provide optimal huA33-C825 and CA doses, followed by additional biodistribution studies ( ¹⁷⁷ Lu-DOTA-Bn doses (˜2.0-111.0 MBq)). To serve as a practical and dosimetric guide for the PRIT study. Furthermore, as described herein, tumors and kidneys were removed 24 hours after injection of ¹⁷⁷ Lu-DOTA-Bn at three different dose levels (11.5, 55.0, and 111.0 MBq) and ¹⁷⁷ by autoradiography ^. The micro-distribution of Lu activity was examined ex vivo, and the correlation between ¹⁷⁷ Lu activity and xenograft and tissue morphology was examined by hematoxylin and eosin staining. Furthermore, the absolute uptake of SW1222 tumors of huA33-C825 (0.25 mg / mouse) 24 hours after injection estimated based on radiotracer studies with ¹³¹ I-huA33-C825 was ˜90 pmol / g / tumor. Thus, the present invention provides an estimate if one molecule of huA33-C825 has the ability to bind to two molecules of ¹⁷⁷ Lu-DOTA-Bn (thus the maximum binding performance of ¹⁷⁷ Lu-DOTA-Bn is 180 pmol / g tumor) The maximum occupancy is (11 pmol ¹⁷⁷ Lu-DOTA-Bn / 180 pmol = 0.061 or ˜6%). Thus, the present invention has been shown to be particularly effective without resulting in the accompanying harmful radiation response. Furthermore, the present invention particularly relates, in at least some embodiments, to an improved multi-step PRIT method (anti-A33 / anti-DOTA-Bn (metal) bispecific antibody designated huA33-C825), dextran-based CA and ¹⁷⁷ Using Lu-DOTA-Bn), immunodeficient mice with subcutaneously established human colorectal tumor xenografts can be cured with minimal toxicity to normal tissues (including bone marrow and kidney) It shows that.
SW1222 stands out as a relatively well differentiated and vascularized tumor among human colorectal cancers that have been reviewed so far (eg LS174T). Although permitting a homogeneous distribution of target-derived antibodies (Emir et al., 2007, Cancer Res. 15; 67 (24): 11896-11905), these tumors are also relatively radiation resistant. As described herein, the A33 antigen is highly expressed in more than 95% of human colon cancer, with normal expression limited and little excreted into the circulation. In human colorectal cancer, clinical therapeutic target induction to the A33 antigen has been unsuccessful so far. The bispecific antibodies described herein exhibit an affinity for affinity to A33 and DOTA-Bn (metal), which is associated with tumor uptake of radiolabeled lutetium (eg ¹⁷⁷ Lu) and A33 positive tumors Promotes the successful delivery of targeted guided radioimmunotherapy. Furthermore, the bispecific binding proteins utilizing the humanized A33 antibodies described herein have the ability of bivalent binding to A33 and bivalent binding to DOTA-Bn, which includes A33 ⁺ This results in enhanced tumor killing performance and enhanced safety due to lack of catastrophic radiation response. Thus, the PRIT strategy utilizing the bispecific binding protein format described herein is a unique approach for enhancing tumor killing, reducing adverse effects, and treating severe A33 positive cancer To be a powerful remedy for.

In particular, the present invention encompasses the recognition that multispecific binding agents (especially bispecific binding agents such as bispecific antibodies) are particularly useful and / or effective in promoting cell killing. In particular, the present invention demonstrates that the activity of multivalent binding factors that specifically bind both target cell-associated epitopes (eg, tumor antigens) and small molecule haptens (eg, DOTA-Bn (metal)) is an effective immunotherapy for colon cancer. Indicates that it is possible.
For example, in some embodiments of the invention, the multivalent binding agent specifically binds a tumor cell associated epitope and a small molecule hapten. According to such embodiments, the multivalent binding agent can promote binding of the agent to one or both of its target epitopes and / or is mediated by radioimmunotherapy via a small molecule hapten. Target tumor cell killing can be enhanced.
In some embodiments, target cells that are killed include, for example, cells that express a tumor antigen (eg, an A33 positive tumor). Those skilled in the art will recognize suitable target epitopes on cells to which the multivalent binding agents described herein bind as desired.

Nucleic acid constructs and expression The humanized antibodies and multispecific binding agents described herein (eg, bispecific antibodies) can be made using molecular biology methods known in the art. The nucleic acid molecule is inserted into a vector capable of expressing the fusion protein when introduced into a suitable host cell. Suitable host cells include, but are not limited to, bacterial, yeast, insect and mammalian cells. Any of the methods known to those skilled in the art for inserting DNA fragments into vectors can be used to construct expression vectors encoding the fusion proteins of the invention under the control of transcriptional / translational control signals. These methods include in vitro recombinant DNA and synthetic techniques and in vivo recombination (see, eg, Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory; Current Protocols in Molecular Biology, Eds. Ausubel, et al, Greene Publ. Assoc., Wiley-Interscience, NY).
Expression of the nucleic acid molecule according to the present invention can be regulated by a second nucleic acid sequence such that the molecule is expressed in a host transformed with the recombinant DNA molecule. For example, the expression of the nucleic acid molecules of the invention can be controlled by promoter and / or enhancer elements known in the art.
Nucleic acid constructs include regions encoding multispecific binding proteins made from antibodies and / or antibody components. Typically, such multispecific binding proteins will be made from the _VH and / or _VL regions. After identifying and screening for antibodies that exhibit the desired binding and / or functional properties, the variable regions of each antibody are isolated, amplified, cloned, and sequenced. Modifications can be made to the _VH and / or _VL nucleotide sequences. Such modifications include the addition of nucleotide sequences that encode amino acids and / or retain restriction sites, deletions of nucleotide sequences that encode amino acids, or substitutions of nucleotide sequences that encode amino acids. Antibodies and / or antibody components can be made from human antibodies, humanized antibodies or chimeric antibodies.
The nucleic acid construct of the present invention is inserted into an expression vector or viral vector by methods known in the art, and the nucleic acid molecule is operably linked to expression control sequences.

Where appropriate, humanized antibodies and multispecific binding agents described herein can be modified to include codons that are optimal for expression in a particular cell type or organism (eg, US Pat. No. 5,670,356). No. and US Pat. No. 5,874,304). The codon optimized sequence is a synthetic sequence, preferably the same polypeptide (or a biologically active fragment of the full-length polypeptide) as the polypeptide encoded by the parent polynucleotide that is not codon optimized. , Which has substantially the same activity as the full-length polypeptide). In some embodiments, the coding region of the genetic material encoding the antibody component is optimized, in whole or in part, to the codon usage of a particular cell type (eg, eukaryotic or prokaryotic). Can contain modified sequences. For example, the coding sequences for the humanized heavy chain (or light chain) variable regions described herein can be optimized for expression in bacterial cells. Alternatively, the coding sequence can be optimized for expression in mammalian cells (eg, CHO). Such a sequence can be referred to as a codon optimized sequence.
An appropriate host cell is transformed with an expression vector containing the nucleic acid molecule to produce the protein encoded by the nucleic acid construct. Exemplary host cells include prokaryotic cells (eg, E. coli) and eukaryotic cells (eg, COS or CHO cells). Host cells transformed with the expression vector are grown under conditions that allow production of the humanized antibody or multispecific binding factor of the invention, followed by recovery of the humanized antibody or multispecific binding factor.
The humanized antibodies and / or multispecific binding agents of the invention can be purified by any technique, which allows the subsequent formation of stable antibodies or binding agent molecules. For example, without being bound by theory, antibodies and / or multispecific binding factors can be recovered from cells as soluble polypeptides or inclusion bodies, from which antibodies and / or multispecific binding factors are quantified by 8M guanidine hydrochloride. Extracted and dialyzed. Conventional ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography or gel filtration can be used for further purification of the antibodies and / or multispecific binding agents of the invention. The humanized antibodies or multispecific binding agents of the invention can also be recovered from the conditioned medium following secretion by eukaryotic or prokaryotic cells.

Screening and Detection Methods The humanized antibodies or multispecific binding agents of the present invention can also be used to detect and / or measure one or more activities (eg, apoptosis or cell proliferation) of one or more cells. It can be used in in vitro or in vivo screening methods. Screening methods are well known in the art and include cell-free assays, cell-based assays and animal assays. In vitro assays can be in solid phase or in a soluble state, and target molecule detection can be detected by a number of methods known in the art. The above involves the use of a label or detection group (which binds to a target molecule (eg, a cell surface antigen)) that can identify a humanized antibody or multispecific binding agent. Detectable labels can be used with assays using the humanized antibodies or multispecific binding agents of the invention.

Therapeutic agents The humanized antibodies or multispecific binding agents of the present invention can be used as therapeutic agents. In some embodiments, as will be understood in the art, the above is utilized without further modification. In some embodiments, they can be incorporated into the compositions or formulations described herein. In some embodiments, they can be chemically linked or linked (eg, complexed) with one or more agents or entities, eg, payload.
A variety of techniques for conjugating antibody drugs or components thereof with other parts or entities are well known in the art and can be utilized in accordance with the present invention. As an example, radiolabeled antibody agents can be generated according to techniques well known in the art.
For example, monoclonal antibodies can be iodinated by contacting with the following substances: sodium and / or potassium iodide and chemical oxidizing agents (eg sodium hypochlorite) or enzyme oxidizing agents (eg lactoperoxidase). Antibody drugs can be labeled with technetium-99m by a ligand exchange process. The process is performed, for example, by reducing pertechnetate with a stannous solution, chelating the reduced technetium with a Sephadex column, and applying the antibody to this column. In some embodiments, provided antibody agents use direct labeling techniques to incubate, for example, pertechnetate, a reducing agent (eg, SNCl ₂ ), a buffer solution (eg, sodium-potassium phthalate solution) and an antibody. Be labeled by A mediating functional group (present as a metal ion) that is often used to couple radioisotopes with antibodies is diethylenetriaminepentaacetic acid (DTPA), or ethylenediaminetetraacetic acid (EDTA), or 1,4,7,10-tetra Azacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or p-aminobenzyl-DOTA (DOTA-Bn). Radioisotopes can be detected, for example, by dosimetry.

The ability of the humanized antibodies or multispecific binding agents of the invention to show high affinity binding to one of the target antigens makes them therapeutically effective to induce targets in target antigen-expressing cells. To make it useful. Thus, in some embodiments, the affinity of a humanized antibody or multispecific binding agent for one target antigen is enhanced and said multispecific binding agent (or Fc receptor in the case of a humanized antibody). It may be desirable not to increase the affinity for other target antigens to which the body is bound. For example, in the context of tumor killing, it may be beneficial to increase or decrease affinity for a tumor antigen under certain conditions, but not increase or decrease affinity for a second antigen. Thus, the use of a humanized antibody or multispecific binding factor as described herein enhances the binding affinity of the humanized antibody or multispecific binding factor for a tumor antigen in a patient having a tumor that expresses the tumor antigen. It can be beneficial to do.
The present invention provides humanized antibodies and / or multispecific binding agents described herein as therapeutic agents for treating patients with tumors that express an antigen to which such multispecific binding agents can bind. provide. Such humanized antibodies and / or multispecific binding agents can be used in methods of treating or diagnosing the human and animal body.

Administration The present invention provides a method of administering to a subject animal in need of treatment an effective amount of a therapeutically active agent (eg, a humanized antibody and / or multispecific binding agent) as described herein.
The humanized antibodies or multispecific binding agents described herein can be administered by a variety of methods known in the art for therapeutic delivery of drugs (eg, proteins or nucleic acids). Using such a method, a humanized antibody or multispecific binding factor or a nucleic acid encoding the humanized antibody or multispecific binding factor of the present invention can be used for killing or inhibiting the growth of target cells in the subject animal. Can be used. Said methods are, for example, cell transfection, gene therapy, direct administration by delivery vehicle or pharmaceutically acceptable carrier, indirect delivery by providing a recombinant cell comprising a nucleic acid encoding a multispecific binding agent of the invention. .
Various delivery systems are known and can be used to administer the humanized antibody or multispecific binding agent of the invention. These include, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, eg, Wu and Wu, 1987, J. Biol. Chem. 262: 4429-4432), construction of nucleic acids as part of retroviruses or other vectors. The route of administration can be enteral or parenteral and includes, but is not limited to, intravenous, subcutaneous, intramuscular, parenteral, transdermal, or transmucosal (eg, oral or nasal). In some embodiments, the multispecific binding agents of the invention are administered subcutaneously. In some embodiments, the multispecific binding agent is administered with other biologically active agents.
In certain embodiments, therapies using the therapeutically active agents described herein (eg, humanized antibodies or multispecific binding agents) can be combined with other therapies, particularly including other anti-tumor therapies. Those of ordinary skill in the art will readily appreciate upon reading this disclosure. In some embodiments, such other anti-tumor therapies are, for example, one or more chemotherapeutic agents, administration of immunomodulators, radiation therapy, radiofrequency ultrasound therapy, surgery, etc. Including.
In some embodiments, the relative timing of administration with a therapeutically active agent described herein (eg, a humanized antibody or multispecific binding agent) and another therapy in combination is selected to optimize the effect. can do.
To name a few, in some embodiments, a therapeutically active agent described herein is administered for a period of time and sufficient time (eg, according to a dosing regimen) to saturate the tumor cells. In some embodiments, unbound therapeutically active agent is removed from the bloodstream after administration. In some embodiments, such removal is performed (eg, allowed to perform) prior to administration of another factor.

In some specific embodiments, the therapeutically active agents described herein are administered in combination with another agent that targets DOTA-Bn. In some such embodiments, another factor holds the payload. In some embodiments, the payload is a therapeutic drug payload or can include a therapeutic drug payload (eg, a toxic payload). In some implementations, the payload is or can include a detection agent payload.
In some specific embodiments, a therapeutically active agent described herein (eg, a humanized antibody or multispecific binding agent) is administered such that tumor cells are saturated and subsequently targets DOTA-Bn A second factor is administered (and holds the payload). Optionally, at least one third factor is administered that targets DOTA-Bn (eg, can hold a different payload).
In some embodiments, the second and optionally third factor is administered after a period of time from administration of the therapeutically active agent described herein, which period allows for the disappearance of unbound factor. There may be a sufficient period of time. In some embodiments, the second and optionally third agent is administered without further administration of the therapeutic agent. For example, in some embodiments, a therapeutically active agent described herein is administered according to a regimen comprising at least one cycle of: (i) administration of the therapeutic agent (sometimes saturating the appropriate tumor cells) (Ii) a second and optionally at least one third factor (eg a factor capable of targeting DOTA-Bn and optionally holding a payload); optionally a second and And / or additional administration of the third factor, but no additional administration of the therapeutic factor. In some embodiments, a therapeutic regimen can include a plurality of such cycles. In some embodiments, the regimen can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 cycles or more.
In some embodiments, the therapeutic regimen includes only one single cycle that includes administration of the therapeutic agent. In some embodiments, such treatment regimens can include one or more cycles comprising step (ii) and optionally step (iii), but without additional administration of the therapeutic agent.
In some embodiments, the pioneering administration of a therapeutic agent described herein is such that the agent with which the therapeutic agent is combined is administered alone (ie, administration without pioneering administration of the therapeutic agent described herein). Allows combination therapies that exhibit a therapeutic index broader than the therapeutic index. In some embodiments, such a wider therapeutic index is improved by at least a log conversion fold.

Pharmaceutical Compositions The present invention further provides pharmaceutical compositions, which comprise a humanized antibody or multispecific binding agent of the present invention and a pharmaceutically acceptable carrier or excipient. The composition can also include one or more additional substances that are therapeutically active if desired.
The description of the pharmaceutical composition provided herein is basically aimed at pharmaceutical compositions suitable for prescription administration to humans, but in general such compositions are intended for all types of animals. One skilled in the art will appreciate that it is also suitable for administration. In order to make the composition suitable for administration to a variety of animals, it is well known to modify a pharmaceutical composition suitable for administration to humans, and veterinary pharmacologists usually simply Such modifications can be designed and / or implemented through routine experimentation.
Formulations of the pharmaceutical compositions described herein can be prepared by any method known or later developed in the pharmacology field. In general, such preparative methods involve the step of associating the active ingredient with a diluent or another excipient and / or one or more other accessory ingredients, followed by where necessary and / or desired. Involves shaping the product and / or packing it into the desired single dose or multi dose unit.
The pharmaceutical compositions of the invention are prepared, packaged and / or sold in bulk as a single unit dose and / or as multiple single dose units. As used herein, a “unit dose” is a discrete amount of a pharmaceutical composition that contains a predetermined amount of an active ingredient. The amount of the active ingredient is generally determined according to the dosage of the active ingredient administered to the subject animal and / or an easy-to-use portion of such dosage (eg 1/2 or 1/3 of such dosage). )be equivalent to.
The relative amounts of the active ingredients, pharmaceutically acceptable excipients, and / or any additional ingredients in the pharmaceutical composition of the invention will depend on the entity, size and / or condition of the subject animal being treated. In addition, it will vary depending on the route to which the composition is administered. By way of example, the composition may contain from 0.1% to 100% (w / w) active ingredient.

The pharmaceutical formulation may additionally comprise pharmaceutically acceptable excipients, including any and all solvents, dispersion media, diluents, or other as used herein Liquid vehicles, dispersion or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like are included as appropriate for each desired dosage form. The following references disclose various excipients used in the formulation of pharmaceutical compositions and known techniques for their preparation: Remington's The Science and Practice of Pharmacy, 21st Edition, AR Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006); the foregoing is hereby incorporated by reference. Any substance or derivative thereof, such as by any conventional excipient, for example by causing undesirable biological effects or otherwise interacting in a deleterious manner with any other component of the pharmaceutical composition Unless otherwise specified, such use is intended to be within the scope of the present invention.
In some embodiments, the pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% pure. In some embodiments, the excipient is approved for human or veterinary use. In some embodiments, the excipient is approved by the US Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopeia (USP), the European Pharmacopeia (EP), the British Pharmacopeia, and / or the International Bureau of Law.
Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include inert diluents, dispersion and / or granulating agents, surfactants and / or emulsifiers, disintegrants, binders, preservatives, Includes but is not limited to buffering agents, lubricants, and / or oils. Such excipients can optionally be added to the pharmaceutical formulation. Excipients such as coconut butter and suppository waxes, colorants, coatings, sweeteners, flavors, and / or fragrances may be present in the composition according to the judgment of the dispenser.
General considerations in prescribing and / or manufacturing medical factors can be found, for example, in the following literature: Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (which is hereby incorporated by reference). include).

Kits The present invention further provides a pharmaceutical pack or kit comprising one or more containers filled with at least one humanized antibody or multispecific binding agent (eg, bispecific antibody) as described herein. The kit can be used in any application method including, for example, treatment or diagnosis. In some cases, such containers may be accompanied by a notice of a government-designated form that regulates the manufacture, use or sale of the medicinal product or biological product (the notice may be (a) manufactured, for human administration) Used or sold by the supervisory authority, (b) instructions for use, or both).
Other features of the present invention will become apparent in the course of describing the following exemplary embodiments. The foregoing embodiments are provided for illustration of the invention and are not intended to limit the invention.
The following examples are provided to illustrate those skilled in the art how to make and use the methods and compositions of the invention and are not intended to limit what we consider to be their invention. . Unless otherwise indicated, temperatures are given in degrees Celsius and pressures are at or near atmospheric.

In vitro characterization of huA33-C825 In particular, the present invention encompasses the insight that humanized antibody A33 (huA33) is particularly important for the construction of multispecific binding factors (eg bispecific antibodies) . Without being bound by any particular theory, we have found that sub-optimal tumor doses and therapeutic indices observed with radiolabeled monospecific huA33 (eg ¹³¹ I-huA33) are multispecific It was proposed that it could be overcome by using huA33.
In this example, the first antigen-binding site based on the humanized antibody A33 and a small molecule hapten (for example, benzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) The preparation of a bispecific antibody composed of a second antigen binding site that binds (DOTA-Bn)) is described. The data provided herein demonstrates successful production of a bispecific antibody (referred to as A33-C825) that targets colorectal cancer cells. As described herein, an anti-DOTA-Bn single chain Fv fragment (ScFv) based on affinity matured 2D12.5 antibody was ligated to the carboxy terminus of the humanized A33 light chain. The major drawbacks of the development of preliminary target-guided radioimmunotherapy (PRIT) were normal tissue overexposure, immunogenicity, suboptimal tumor dose and low therapeutic index. As shown below, the bispecific antibodies of the present invention overcome such deficiencies and provide an effective PRIT feasibility against cancers expressing human A33 antigen (eg, colorectal cancer).
An exemplary biochemical purity analysis of huA33-C825 by SE-HPLC is shown in FIG. SE-HPLC showed several minor peaks (presumed to be aggregates that could be removed by gel filtration) along with a major peak (90% by UV analysis) with a MW of approximately 210 KDa. The bispecific antibody showed stability by SE-HPLC and Biacore after multiple freeze-thaw cycles. Binding affinity was measured by Biacore T100. Exemplary results are shown in Table 3. An exemplary sensorgram is shown in FIG.

As shown in this embodiment, huA33-C825 showed lower affinity for comparison with human A33 antigen and monospecific HuA33 antibodies (K _D is 63.5nM versus 1.71 nm, Figure 2A). huA33-C825 is BSA- (Y) compared to a control bispecific antibody that has a first antigen binding site that does not bind to the A33 antigen and a second antigen binding site that binds DOTA-Bn (metal). maintaining high binding affinity for -DOTA-Bn (K _D is 11.6nM versus 21.2 nm, Figure 2B). Taken together, this example demonstrates the construction of bispecific antibodies that bind to human A33 antigen and small molecule haptens (eg, DOTA-Bn) and maintain high affinity for both targets. Furthermore, the reduced affinity for human A33 antigen observed with huA33-C825 provides more rapid clearance compared to the parent huA33-IgG1 antibody.

Optimization of PRIT with huA33-C825, dextran remover (dextran-CA) and ¹⁷⁷ Lu-DOTA-Bn This example demonstrates the multispecific binding factor for preliminary target-guided radioimmunotherapy (PRIT) of A33 positive tumors ( For example, the effectiveness of bispecific antibodies). In particular, this example uses a bispecific antibody as described in Example 1 as a function of the amount of scavenger (CA), using a pre-targeted induced radioimmunity (PRIT) protocol in SW1222 cancer-bearing rodents. The optimization of tumor target induction is described. As shown below, a progressive increase in therapeutic index is observed with increasing dose, but no decrease in absolute tumor uptake is observed.
Biodistribution of SW1222 tumor-bearing mice 24 hours after ¹⁷⁷ Lu-DOTA-Bn injection using a fixed ratio of 0.1-0.6 mg huA33-C825 (0.48-2.86 nmol) and CA to ¹⁷⁷ Lu-DOTA-Bn (5.6 MBq) Based on a pilot study (with 0.25-0.6 mg of huA33-C825, the tumor ¹⁷⁷ Lu-activity concentration showed a plateau of -15-18% ID / g), a 0.25 mg / mouse dose of huA33-C825 was selected . Additional biodistribution experiments were then performed to optimize the CA dose in PRIT using 0.25 mg (1.19 nmol) as the huA33-C825 dose. Tumor-bearing mouse groups (n = 3-4 / group) were injected with huA33-C825 followed by 24 hours later saline (ie vehicle), 2.4% (% to 0.25 mg huA33-C825, w / w) 5% (w / w), 10% (w / w) or 25% (w / w) CA dose (0-62.5 μg / mouse) was injected. Four additional hours later, mice were injected with 5.6 MBq of ¹⁷⁷ Lu-DOTA-Bn and sacrificed for biodistribution analysis 24 hours later. An exemplary optimization of the remover is shown in FIGS. 3A-3D. Exemplary ¹⁷⁷ Lu-DOTA-Bn activity in SW1222 tumors and various normal tissues from a group of mice administered a 25% (w / w) dose of CA is shown in FIGS. 4A-4D.
As expected, CA administration had a significant effect on circulating (ie blood) ¹⁷⁷ Lu-activity (˜8 to 0.1% ID / g for 25% (w / w) from saline (no CA) to 25% (w / w), respectively) ). In addition, the CA dose appears to reduce the tumor's subsequent ability to uptake ¹⁷⁷ Lu-DOTA-Bn. The highest CA dose tested (25%) was considered optimal. This is because the highest radiosensitive tissue (blood and kidney) tumor to normal tissue ratio was highest compared to lower CA dose (but ¹⁷⁷ Lu compared to PRIT with saline). -At the expense of reduced tumor uptake of DOTA-Bn (~ 50% lower uptake)). In particular, ¹⁷⁷ Lu-DOTA-Bn tumor uptake (as% ID / g, mean ± standard error of arithmetic mean) was 17.51 ± 0.90 for saline and CA 65 μg dose levels (25% w / w), respectively ( n = 3) and 8.46 ± 3.74 (n = 4). For saline, the blood, kidney and muscle tumor-to-organ ratios were 2.2 ± 0.4, 4.9 ± 0.6, and 23.2 ± 3.8, respectively. For CA, at 65 μg CA dose level (25% w / w), the blood, kidney and muscle tumor-to-organ ratios were 105.8 ± 52.3, 18.4 ± 13.4, and 282.1 ± 208.7, respectively. Next, a dose titration study of ¹⁷⁷ Lu-DOTA-Bn was performed using the optimized PRIT dose of huA33-C825 and CA. For dose titration study of ¹⁷⁷ Lu-DOTA-Bn, tumors and critical selection tissues (blood, liver, spleen and kidney) the biodistribution data for ¹⁷⁷ Lu-activity,% between ¹⁷⁷ Lu-DOTA-Bn dose group A comparison was made for both ID / g and absolute uptake (kBq / g; see FIGS. 4A and 4B). Finally, a single point biodistribution experiment was performed in SW1222 tumor-bearing mice 24 hours after injection of ¹³¹ I trace-labeled huA33-C825 (0.39-0.40 MBq combined with 1.19 nmol of cold huA33-C825). The absolute antibody uptake (as pmol / g) of huA33-C825 in tumors during PRIT was estimated. Exemplary aggregate values are shown in Table 4 (data presented as arithmetic mean ± SD). An exemplary tumor uptake calculation is shown in FIG. 4D.
As shown in this example, the ¹³¹ I-huA33-C825 uptake (mean ± standard deviation) of the tumor was 3.71 ± 0.97% ID / g. This corresponds to an absolute huA33-C825 uptake of 44 pmol / g (88 pmol / g for ~ 50% immune response ratio).

Calculation of biodistribution and absorbed dose The huA33-C825 described in the preceding examples was tested for its in vivo efficacy. The biodistribution and absorbed dose estimates of radiolabeled DOTA-Bn were determined in SW1222 tumor cell transplanted mice.
In this example, in a group of A33 positive SW1222 tumor-bearing mice, PRIT was performed using optimal doses of huA33-C825 and CA, followed by 2.0 MBq (~ 10 pmol) of ¹⁷⁷ Lu-DOTA-Bn, Further biodistribution studies were performed from 2 to 120 hours after injection of ¹⁷⁷ Lu-DOTA-Bn to determine the residence time of ¹⁷⁷ Lu-activity in tumors and various normal tissues.
In short, the optimal A33-C825 (0.25 mg / mouse) and dextran-removing agent dose (25% (w / w), 62.5 μg) and 2.0 MBq (˜10 pmol) ¹⁷⁷ Lu-DOTA-Bn PRIT Subsequent biodistribution assays were used to determine ¹⁷⁷ Lu-activity of tumors and various normal tissues. A group of SW1222 tumor-bearing mice (n = 4 to 5) was administered 250 μg huA33-C825 followed by 25% (w / w) (62.5 μg) dextran-removal agent 24 hours later Four hours later, 2.0 MBq (˜10 pmol) ¹⁷⁷ Lu-DOTA-Bn was administered. For biodistribution analysis, a group of animals was sacrificed at 2, 24 and 120 hours after injection of ¹⁷⁷ Lu-DOTA-Bn. These data were used as described in Materials and Methods to approximate the absorbed dose for radioimmunotherapy with ¹⁷⁷ Lu-DOTA-Bn (Table 5).
For tumors, ¹⁷⁷ Lu uptake occurred very rapidly following administration, averaging 7.0% ID / g 2 hours after injection. Maximum tumor uptake was 8.5% ID / g at 24 hours, and then decreased to approximately half of 4.0% ID / g at 120 hours after injection after 96 hours. Peak uptake of kidney, liver, spleen and blood was observed 2 hours after injection (mean values were 0.87, 0.70, 0.92 and 0.75% ID / g, respectively), and mean values were 0.27% ID / g (peak of each) Compared to uptake, it decreased to 3.2 times), 0.30% ID / g (down 2.3 times), 0.32% ID / g (down 2.9 times), and 0.02% ID / g (down 37.5 times).
Exemplary estimates of tumor and selected normal tissue absorbed doses for PRIT (including optimal huA33-C825 and dextran remover doses) in female athymic mice with A33 positive colorectal carcinoma subcutaneously are given in Table 6. Indicated. For each target area, the absorbed dose was calculated as the product of the non-intrusive (ie beta) ¹⁷⁷ Lu equilibrium dose constant and the target area's ¹⁷⁷ Lu cumulative activity (assuming that the local absorption of the ¹⁷⁷ Lu beta line is complete) Gamma rays and non-self dose contributions were ignored).

As shown in Table 6, the estimated absorbed dose (as cGy / MBq) of ¹⁷⁷ Lu-DOTA-Bn for blood, tumor, liver, spleen and kidney was 0.9, 65.8, 6.3, 6.6 and 5.3, respectively. Furthermore, in the case of single cycle treatment, the therapeutic index of the tumor is 73 for blood and 12 for the kidney, indicating the cumulative breadth of tumor target induction and no significant toxicity is expected. In fact, no toxicity was observed until day 140 in subjects that showed an overlooked response.
Tumor uptake after PRIT as determined by PET imaging showed results similar to the biodistribution assay described above for both ⁸⁶ Y-DOTA-Bn and ¹⁷⁷ Lu-DOTA-Bn isotypes (data not shown) ).

In Vitro Treatment Test This example illustrates the in vivo efficacy of the huA33-C825 bispecific antibody in mediating tumor burden reduction in mice bearing A33 positive cancer cells. In particular, this example describes the effects of one-cycle and two-cycle therapy on tumor burden in SW1222 tumor-bearing mice.
In the first therapy trial, 5 groups of tumor-bearing mice (n = 6 to 8 / group) were treated with any of the following: vehicle (ie, untreated, n = 8, TV ₇ ; 76 ± 15 mm ³ ) 33.3 MBq ¹⁷⁷ Lu-DOTA-Bn alone (bispecific antibody and vehicle administered at the time of CA injection, n = 6, TV ₇ ; 116 ± 23 mm ³ ), single cycle IgG-C825 PRIT + 33.3 MBq ¹⁷⁷ Lu- DOTA-Bn alone (administered IgG-C825 instead of nshuA33-C825, n = 8, TV ₇ ; 100 ± 10 mm ³ ), or ¹⁷⁷ Lu-DOTA with one cycle huA33-C825 PRIT + 11.1 MBq or 33.3 MBq -Bn (n = 8 for both, TV ₇ is 103 ± 17mm ³ and 93 ± 15mm ^{3 respectively} ). The approximate absorbed dose of the tumor for one cycle huA33-C825 PRIT + 11.1 MBq or 33.3 MBq of ¹⁷⁷ Lu-DOTA-Bn was 730 and 2190 cGy, respectively (from the absorbed dose estimates in Table 6). We observed that the relative tumor uptake decreased as the ¹⁷⁷ Lu-DOTA-Bn dose increased during treatment, indicating that this is approaching possible saturation in the tumor. unknown. This may affect the absorbed tumor dose estimate. Approximate 7% ID / g when used for tumor peak uptake after PRIT + 33.3MBq ¹⁷⁷ Lu-DOTA-Bn dose (ie, lower relative tumor uptake at higher ¹⁷⁷ Lu-DOTA-Bn dose) ), An estimated tumor absorbed dose of ~ 1800 cGy may be more accurate, and an overall effective dose range of 1800-2200 cGy is proposed. An exemplary tumor response (expressed as tumor volume (mm ³ )) for each ¹⁷⁷ Lu-DOTA-Bn treatment group of mice is shown in FIG. Tumor-bearing mice that received no treatment or treatment (consisting of 33.3 MBq ¹⁷⁷ Lu-DOTA-Bn alone or one cycle of IgG-C825 PRIT + 33.3 MBq ¹⁷⁷ Lu-DOTA-Bn) showed no tumor response. ^The latter two groups of scintigraphy administered ¹⁷⁷ Lu-DOTA-Bn showed minimal activity in the tumor area. In contrast, groups treated with one cycle of huA33-C825 PRIT + 11.1 MBq or 33.3 MBq ¹⁷⁷ Lu-DOTA-Bn showed a slight delay in tumor growth up to 15 days after treatment, but CR occurred. There wasn't. For comparison, 23 days after tumor inoculation (16 days after ¹⁷⁷ Lu-DOTA-Bn injection), tumor volume (as mean ± SEM) was untreated, 33.3 MBq ¹⁷⁷ Lu-DOTA-Bn alone, single cycle IgG -C825 PRIT + 33.3 MBq ¹⁷⁷ Lu-DOTA-Bn, or 1 cycle huA33-C825 PRIT + 11.1 MBq or 33.3 MBq ¹⁷⁷ Lu-DOTA-Bn, 1398 ± 206 (n = 8), 1051 ± 167 (n = 5), 877 ± 109 (n = 7), 694 ± 138 (n = 8), and 495 ± 76 (n = 8). Within 30 days after tumor inoculation, the average tumor size for all groups was greater than 1250 mm ³ and the study was completed. Similar results were observed with one cycle huA33-C825 PRIT + ¹⁷⁷ Lu-DOTA-Bn treatment at higher doses with 111.1 MBq ¹⁷⁷ Lu-DOTA-Bn (data not shown).
In the second therapy trial, two-cycle huA33-C825 PRIT treatment was reviewed. Exemplary tumor responses (expressed as tumor volume (mm ³ )) of mice that received two-cycle treatment are shown in FIGS. 6A-6D.
When mice did not receive any treatment (n = 5, TV ₁₀ : 314 ± 77 mm ³ ), all tumors needed to be sacrificed within 30 days due to tumor growth, reaching 500 mm ³ The duration was 13 ± 2 days. 2/5 PRIT + 11.1 MBq ¹⁷⁷ Lu-DOTA-Bn (total ¹⁷⁷ Lu-DOTA-Bn dose is 22.2 MBq, estimated tumor dose is 1460 cGy) (n = 5, TV ₁₀ 462 ± 179 mm ³ ) Of animals showed CR (Figure 6A). In recurrent mice, the period of reaching 500 mm ³ was 9 days (TV ₁₀ : 391 mm ³ ) or 36 days (TV ₁₀ : 712 mm ³ ). 2 cycles of PRIT + 33.3 MBq ¹⁷⁷ Lu-DOTA-Bn (total dose of ¹⁷⁷ Lu-DOTA-Bn is 66.6 MBq, estimated tumor dose is 3600-4400 cGy) (n = 5, 344 ± 105 mm ³ ) is 5/5 The animals produced CR (Figure 6B). In these recurrent tumors, the period of reaching 500 mm ³ is 12 days (TV ₁₀ : 325 mm ³ ), 65 days (TV ₁₀ : 502 mm ³ ), 7 days (TV ₁₀ : 341 mm ³ ) and 23 days (TV ₁₀ : At 345 mm ³ ), only one mouse had a tumor size of less than 10 mm ³ when sacrificed. 2 cycles of PRIT + 55.5 mCi ¹⁷⁷ Lu-DOTA-Bn (total dose of ¹⁷⁷ Lu-DOTA-Bn is 111.0 MBq; approximate tumor dose is 2580 cGy (based on peak tumor uptake of 3% ID / g)) (N = 4, 236 ± 54 mm ³ ) produced CR in 4/4 animals (FIG. 6C). These recurrent tumors period reached 500 mm ^3, the 34 days ^{_{(TV 10: 295mm 3),}} 45 days (TV _10: 263mm ³⁾ and 42 days: in (TV ₁₀ 175mm ^3), only one mouse When sacrificed, the tumor size was 44 mm ³ . After treatment with 2 cycles of PRIT + 33.3 mCi ¹⁷⁷ Lu-DOTA-Bn (total dose of ¹⁷⁷ Lu-DOTA-Bn was 66.6 MBq), the mean recurrence to 500 mm ³ was 27 ± 26 days. Treatment with 2 cycles of PRIT + 55.5 mCi ¹⁷⁷ Lu-DOTA-Bn (total dose of ¹⁷⁷ Lu-DOTA-Bn was 111 MBq) had a mean recurrence to 500 mm ³ of 40 ± 6 days. Estimates of absorbed radiation dose (expressed in Gy) for each treatment regimen are shown in Table 7.

A similar trend for survival was observed in animals after 140 days of treatment (data not shown).

Toxicity This example illustrates the in vivo toxicity of the humanized A33 bispecific antibody described in the previous examples.
Briefly, the sum treated with either 2 cycles PRIT + 11.1 MBq ¹⁷⁷ Lu-DOTA-Bn (n = 3) or 2 cycles PRIT + 1.5 mCi ¹⁷⁷ Lu-DOTA-Bn (n = 3) Six mice were subjected to renal, bone marrow, liver and splenic pathological anatomical assessments up to 9 weeks after treatment. 2 cycles 3/5 mice showed no CR after treatment with ¹⁷⁷ Lu-DOTA-Bn of PRIT + 0.3mCi of, voted second ¹⁷⁷ Lu-DOTA-Bn dose injection (i.e. from the treatment) after 5 days It was attached to. These mice did not show any reduction in tumor size after treatment and needed to be sacrificed due to excessive tumor burden. 3/3 mice showed normal kidney and bone marrow and no radiation-induced injury. One third of the mice showed extramedullary hematopoiesis and the other two animals in this group had normal liver. For 1/3 of the mice, the spleen (white pulp) showed follicular lymphocyte hyperplasia, and the spleen was normal in the other two animals in this group. In mice treated with 2 cycles of PRIT + 55.5 MBq, only one mouse was evaluated 7 weeks after treatment, while the other 2 were evaluated 9 weeks after treatment. All three of these mice showed CR, after which the tumor recurred and needed to be sacrificed for excessive tumor burden. In 3/3 mice, kidney, bone marrow and liver were normal. In 1/3 of the mice, the spleen (white pulp) showed follicular lymphocyte hyperplasia, and the spleen was normal for the other 2 animals in this group.
In particular, this example confirms that huA33-C825 effectively reduces tumor burden in vivo (ie reduces tumor growth) and provides effective PRIT.

Theranostic PRIT with healing effect
This example describes the use of the humanized A33 bispecific antibodies described in the previous examples and, among other things, demonstrates that treatment with these antibodies can have a curative effect. In particular, this example shows a therapeutic regimen for therapeutic diagnosis and having a curative effect. The regimen included additional treatment cycles that increased the total dose activity.
Nude mice with SW1222 subcutaneous colonized xenografts (n = 20, tumor volume = 102 ± 40 mm ³ (mean ± standard deviation (SD)) were treated as follows (n = 5-10 / group): no treatment (N = 5), ¹⁷⁷ Lu-DOTA-Bn only (n = 5), or 3-cycle PRIT regimen consisting of anti-GPA33 PRIT + 55 MBq ¹⁷⁷ Lu-DOTA-Bn (n = 10, 165 MBq total) Continuous nanoSPECT / CT Imaging was performed on 5 randomly selected mice (the mice received DPRIT for up to 160 hours after the first cycle injection of ¹⁷⁷ Lu-DOTA-Bn for dosimetric calculations).
DPRIT elicited a complete tumor response in 10/10 mice (10/10 died 21 days after tumor in the control) and 100-day tumor-free survival of all treated animals with no apparent toxicity It was. Necropsy of 5/10 mice at 100 days demonstrated healing and did not show histopathological findings that should be noted in the assessment of kidney, liver, spleen and / or bone marrow (data not shown) ). The estimated dose of ¹⁷⁷ Lu-radiation exposure to the tumor following cycle 1 was 4556 ± 637 rad (mean ± SD, n = 5). Based on these data, the first order approximation of total ¹⁷⁷ Lu-radiation exposure to the tumor following curative DPRIT (ie 3 cycles) is 14000 rad (blood and kidney radiation dose 150 rad, respectively, therapeutic index (TI): 93) and 875 rad (TI: 16).
Lutetium-177 nanoSPECT / CT imaging of 3 cycle PRIT regimen treated mice showed strong contrast of visible uptake of tumor and minimal uptake of background tissue. (Data not shown). The TI was ~ 70: 1. Based on non-invasive in vivo cross-sectional images of living mice, detection of tumors below 10 mg was observed. Particularly in this example, huA33-C825 can effectively reduce tumor burden in vivo, and PRIT-based therapeutic and diagnostic drugs can have a curative effect and / or can be used for detection of small tumors. Is confirmed.

This “real-time” simultaneous theranostic treatment and image-guided dosimetry This example describes the in vivo response to a therapeutic and diagnostic dual-use DOTA-PRIT regimen using the humanized A33 bispecific antibody described in the previous examples. Reveal therapeutic efficacy through concurrent treatment and image-guided dosimetry. In particular, nanoSPECT / CT was used for high resolution quantitative imaging of mice receiving ¹⁷⁷ Lu-DPRIT treatment for “real time” dosimetry.
One SW1222 tumor-bearing nude mouse (volume: 100 mm ³ by caliper measurement) was treated with a single cycle of anti-GPA33 PRIT + 55 MBq of ¹⁷⁷ Lu-DOTA-Bn, and nanoSPECT / CT imaging revealed that ¹⁷⁷ Lu-DOTA- Images were made at 3 time points after Bn injection (1, 24 and 160 hours after injection). Shown in FIG. 8 is a maximum intensity nanoSPECT / CT image of the lower flank where the tumor is present. Images were adjusted with known active standards with injection time corrected decay. The active concentration of the tumor was determined using analysis of the problem area of the adjusted image. In particular, this example confirms that huA33-C825 effectively reduces tumor burden in vivo, and that high-analysis quantitative imaging is one method that can be used to measure efficacy.
Materials and methods for the examples:
Tumor cell lines and cell culture reagents Human colorectal cancer cell line SW1222 was obtained from the following institution (Ludwig Institute for Cancer Immunotherapy, New York, NY) and maintained by serial passage. Cells were cultured in Minimal Essential Medium supplemented with the following in a 37 ° C environment containing 5% CO ₂ : 10% heat-inactivated fetal calf serum, 2.0 mM glutamine, 100 units / mL penicillin and 100 Unit / mL streptomycin. Upon receipt of the cell line, cultures were established and cryopreserved in small aliquots to limit passage to less than 3 months. In addition, the mycoplasma was examined periodically using a commercial kit (Lonza) according to the manufacturer's specifications. A solution of 0.25% trypsin / 0.53 mM EDTA in Hank's buffered salt solution without calcium and magnesium was used for trypsinization at passage and cell harvest.
Cloning and expression of huA33-C825
huA33-C825 is a humanized antibody A33 (huA33; King) using a platform previously described in Cheal et al. (Cheal, SM et al. (2014, Mol. Cancer Ther. 13 (7), page 10). , DJ et al, 1995, British J. Cancer 72:. 1364-1372 .huA33-C825 prepared using the variable regions (V _H and V _L) of) was produced in CHO cells using a mammalian expression vector, Purified by protein A affinity chromatography as described (Cheal et al., Supra).
Exemplary bispecific antibodies of the invention are presented in Table 8 (huA33-C825: humanized A33 IgG1-murine C825 scFv, huA33-huC825: humanized A33 IgG1-humanized C825 scFv). For DNA sequences, leader sequences are presented as underlined text. For amino acid sequences, leader sequences are presented as underlined text, linker sequences are presented as bold text, and variable region sequences are presented as italic text.

Surface plasmon resonance test Biacore T100 biosensor, CM5 sensor chip, and related reagents were purchased from GE Healthcare. Recombinant human A33 protein was purchased from Novoprotein. The BSA- (Y) -DOTA-B conjugate was prepared according to the description (Cheal et al., Supra). A33 and DOTA antigens were immobilized using an amino coupling kit (GE Healthcare). Purified bispecific and control antibodies were analyzed and the data were fitted to a bivalent analyte model using Biacore T100 evaluation software as described (Cheal et al., Supra).
PRIT reagents, protocols and xenograft studies All animal experiments were approved by the Institutional Animal Care and Use Committee of the Sloan Kettering Memorial Cancer Center and followed in-house guidelines for proper and humane use. Athymic nu / nu female mice (6-8 weeks old (Harlan Sprague Dawley)) were acclimated to the rearing box for at least one week. Groups of animals were injected subcutaneously in the left flank with 5 × 10 ⁶ cells formulated 1: 1 with Matrigel (BD Biosciences) A33 positive SW1222. Established tumors (100-900 mm ³ ) were observed at 7-10 days using the formula for the volume of the ellipsoid (V = 4 / 3π (length / 2xwidth / 2xheight / 2)). All reagents were administered intravenously (iv) from the side of the tail vein. The PRIT protocol included the following injections: huA33-C825 [t = -28 hours] followed by CA [t = -4 hours] after 24 hours (CA is dextran- (Y) -DOTA-Bn containing 500 KDa). Combined and prepared as described in Orcutt et al. (Orcutt et al., 2011, Nucl. Med. Biol. 38: 223-233) and formulated in saline for injection (Y ) -DOTA-Bn molar substitution ratio was 61 (Y) -DOTA-Bn / dextran), and after 4 hours ¹⁷⁷ Lu-DOTA-Bn [t = 0 hours] (as previously described , aminobenzyl _{-DOTA (p-NH 2 -Bn-} DOTA) (Macrocyclics) and ¹⁷⁷ LuCl ₃ (specific activity ~30Ci / mg; Perkin Elmer) prepared by incubating, formulated in saline for injection did). In addition, huA33-C825 was trace radiolabeled with I-131 to estimate tumor uptake during PRIT. ¹³¹ I-huA33-C825 was prepared using IODOGEN method (Cheal, S. et al., 2014, Mol. Cancer Ther. 13 (7): 1-10) (final specific activity 95.5 MBq / mg, cold huA33-C825 was added to achieve the desired mg dose (radiochemical purity using size exclusion high pressure liquid chromatography was> 98%), and in vitro cell-bound immunoreactivity was essentially Lindmo's It was determined using SW1222 cells as described in the method (Lindmo, T. et al., 1990, J. Immunol. Meth. 126 (2): 183-189). For PRIT with non-specific IgG-C825, an equivalent mg dose of GD2 target-derived bispecific antibody (hu3F8-C825) was used instead of huA33-C825. For ex vivo biodistribution analysis, mice are euthanized by CO ₂ (gas) asphyxia, tumors and selected organs are collected, washed and air-dried, weighed, and radiated by a gun machine titration measurement (Perkin Elmer Wallac Wizard 3) Potency assay was performed. Measurement rates were corrected for background and decay, converted to activity using system calibration factors, normalized to the activity administered, and expressed as percent injected dose / gram (% ID / g). Differences in ¹⁷⁷ Lu-activity concentrations in tumors and various tissues were analyzed by student independent t-test where appropriate.

Estimated absorbed dose
A group of A33-positive SW1222 tumor-bearing mice (n = 4-5) with 0.25 mg huA33-C825, CA (62.5 μg; 25% (w / w)), and 1.85-2.0 MBq (˜10 pmol) ¹⁷⁷ Lu- DOTA-Bn was administered and sacrificed at 2, 24 and 120 hours after injection. For each tissue, non-disintegrated modified time activity concentration data is analytically integrated using Excel to fit one component, two components, and, where appropriate, more complex exponential functions, and accumulated per unit dose activity An active concentration was obtained. The ¹⁷⁷ Lu equilibrium dose constant for non-invasive radiation (8.49 g-cGy / MBq-h) was used to approximate the tumor-to-tumor and sorted-organ-to-organ self-absorbed doses (with only ¹⁷⁷ Lu beta rays Assuming complete local absorption, gamma and non-self-dose contributions were ignored). To determine sorting tissue (i.e. blood, liver, spleen and kidney) having a tumor and best absorbed dose effects of ¹⁷⁷ Lu-DOTA-Bn amount for relative uptake of ¹⁷⁷ Lu-DOTA-Bn of, SW1222 tumor-bearing Female athymic nude mice (n = 5 / group) were administered the following: 0.25 mg (1.19 nmol) huA33-C825 (t = −28 hours) and 62.5 μg CA (t = −4 hours), followed by Either 11.1MBq (11.14-11.40MBq), 55.5MBq (54.61-55.06MBq), or 111MBq (109.52-112.5MBq). ^For biodistribution analysis of ¹⁷⁷ Lu activity, all groups were sacrificed 24 hours after injection of ¹⁷⁷ Lu-DOTA-Bn (ie, maximum tumor uptake time).
PET imaging of PRIT + ⁸⁶ Y-DOTA-Bn A single group of mice bearing A33 positive SW1222 tumors on the shoulder (n = 5) was treated with 0.25 mg huA33-C825, CA (62.5 μg; 25% (w / w)), and 8.6-8.8 MBq (˜50 pmol) of ⁸⁶ Y-DOTA-Bn and non-invasive using microPET focus 120 (CTI Molecular Imaging, Inc. Knoxville, TN) 2 and 20 hours after injection Image. The following imaging acquisition parameters were used: 350-750 keV energy window, 6 nsec coincidence timing window, and 20 minutes acquisition time. The obtained list mode data was sorted into 2D histograms by Fourier rebinning, and the cross section was reconstructed into a 128x128x95 matrix by filter back projection (the reconstructed spatial analysis is 2.6 mm full width at half maximum (FWHM)). The images were corrected for scanner response heterogeneity, dead time count loss, physical disruption (relative to injection time), and ⁸⁶ Y positron branching ratio. No attenuation, variance, or partial volume averaging correction was applied. Voxel count rates were converted to active concentrations using an empirically determined mouse system calibration factor (ie, μCi / mL / cps / voxel). Subsequently, the resulting image data was normalized to dose activity, and problem area analysis determined the percent injected dose per gram tissue unit (% ID / g) corrected for radioactivity decay with respect to injection time. . Image and problem area (ROI) analysis (maximum ROI, as% ID / g) was performed using AsiPRO VM 5.0 software (Concorde Microsystems, Knoxville, TN). Animals were sacrificed 24 hours after injection for ex vivo biodistribution analysis.

Autoradiography and immunohistochemistry
From sorted mice injected with huA33-C825 PRIT, followed by ¹⁷⁷ Lu-DOTA-Bn 11.1 (11.14-11.40), 55.5 (54.61-55.06), or 111 MBq (109.52-112.5 MBq) (sacrifice 24 hours after injection) The frozen OCT-embedded tumor and kidney were excised as 10 μm sections with a cryostat (Avantik, Springfield, NJ) and immediately exposed to an imaging plate (Fuji Photo Film, Kanagawa, Japan) for 72 hours, after which Typhoon FLA 7000 Scanning was performed using a scanner (GE, Pittsburg, PA). The same sections were stained with hematoxylin and eosin and scanned under an Olympus BX60 microscope (Olympus, Central Valley, PA) with a movement control stage. Both autoradiography and microscopic images were processed and analyzed using ImageJ (NIH).
Treatment and scintigraphic studies Subcutaneous colonized A33 positive SW1222 tumor-bearing mice were treated with huA33-C825 or non-specific (ns) IgG-C825 PRIT (ie single cycle treatment, ¹⁷⁷ Lu-DOTA 7 days after tumor inoculation) -Bn injection), or two cycles of PRIT (ie, a two-cycle treatment study, with ¹⁷⁷ Lu-DOTA-Bn injections performed 10 and 17 days after tumor inoculation). For the two-cycle treatment study, the tumor volume (TV10) at day 10 after tumor inoculation was noted (ie the day of the first ¹⁷⁷ Lu-DOTA-Bn injection) and expressed as mean ± SD when appropriate. The treatment response was described using the following definition: Complete response (CR) is defined as tumor reduction to less than 100 mm ³ . Persistent response (DR) is defined as survival at 140 days after treatment. Excessive tumor burden is defined as greater than 1000 mm ³ . For the scintigraphy study, the A33 positive SW1222 tumor-bearing mouse selection group undergoing treatment was treated with gas before scanning for 30 minutes (~ 10 ⁵ counts / image) with nanoSPECT (Bioscan, Washington DC) 20 hours after injection. Placed under anesthesia by inhalation. The scanning used a low energy high analysis collimator and a 208 keV window setting. Images were reconstructed into a 256x256 matrix using Bioscan HiSPECT software and uploaded to the ASIPro VM for analysis.

In modifying the elements of a claim in an equivalent claim, the terms representing the order, such as “first”, “second”, “third”, etc., are of their own priority, superiority. Or any other claim having the same name as one claim element having a name, but not implying the order of the listed elements relative to another element or the temporal order in which the work of a method is performed. It is simply used as an indicator to distinguish the elements of the claim from each other.
As used herein in the specification and in the claims, the articles “a” and “an” should be understood to include a plurality of corresponding words, unless expressly indicated otherwise. A claim or explanation that includes “or” between one or more members of a group is present in a given product or process that has one, two, or all of the group members If used in a product or process or otherwise associated with the product or process, it meets the requirements unless otherwise indicated or otherwise clear from the context Is considered to be. The present invention includes a plurality of embodiments in which one or more members of the group are present in a given product or process, utilized above or otherwise. And embodiments related to the foregoing. The present invention also includes a plurality of embodiments, in which two or more group members or all group members are present in a given product or process, or are utilized above, Or otherwise related to the above. Furthermore, the invention is intended to enable one or more limitations, elements, sections, descriptive phrases, etc. derived from one or more of the recited claims to be inconsistent or inconsistent unless otherwise indicated. It is understood that all modifications, combinations and permutations introduced in other claims subordinate to the same basic claim (or any other claim as related) are included unless otherwise apparent. It will be appreciated that if an element is listed (eg, in a Markush group or similar format), each subgroup of that element is also disclosed, and any element can be removed from that group. In general, when the invention or features of the invention include specific elements, features, etc., certain embodiments of the invention or features of the invention are derived from such elements, features, etc. Or consist essentially of such elements, features, etc. For the sake of brevity, these embodiments are not individually described herein in many terms in each case. It is understood that any embodiment or feature of the present invention may be explicitly excluded from the claims regardless of whether a specific embodiment or feature exclusion is listed herein. Should. Publications, websites and other reference materials cited herein are included herein by reference to describe the background of the invention and to provide additional details regarding the practice of the invention. It is.
Having described several features of at least one embodiment of the present invention, it should be understood that various changes, modifications and improvements will be readily apparent to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are merely exemplary and the invention is described in detail in the following claims.

References
Ackerman, ME et al., 2008, A33 antigen displays persistent surface expression, Cancer Immunol.Immunother. 57 (7): 1017-1027.
Ackerman, ME et al., 2008, Effect of antigen turnover rate and expression level on antibody penetration into tumor spheroids, Mol. Cancer Ther. 7 (7): 2233-2240.
Barendswaard, EC et al., 1998, Rapid and specific targeting of monoclonal antibody A33 to a colon cancer xenograft in nude mice, International J. Oncol. 12: 45-53.
Carrasquillo, JA et al., 2011, 124I-huA33 Antibody PET of Colorectal Cancer, J. Nucl. Med. 52: 1173-1180.
Cheal, SM et al., 2014, Preclinical Evaluation of Multistep Targeting of Diasialoganglioside GD2 Using an IgG-scFv Bispecific Antibody with High Affinity for GD2 and DOTA Metal Complex, Mol. Cancer Ther. 13 (7): 1-10.
Cheal, SM et al., 2014, Evaluation of glycodendron and synthetically-modified dextran clearing agents for mult-step targeting of radioisotopes for molecular imaging and radioimmunotherapy, Mol. Pharm. 11 (2): 400-416.
El Emir, E. et al., 2007, Predicting Response to Radioimmunotherapy from the Tumor Microenvironment of Colorectal Carcinomas, Cancer Res. 67 (24): 11896-11905.
Goodwin, DA et al., 1994, Pharmacokinetics of pretargeted monoclonal antibody 2D12.5 and 88Y-Janus-2- (p-nitrobenzyl) -1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA) in BALB / c mice with KHJJ mouse adenocarcinoma: a model for 90Y radioimmunotherapy, Cancer Res. 54 (22): 5937-5946.
King, DJ et al., 1995, Preparation and preclinical evaluation of humanised a33 immunoconjugates for radioimmunotherapy, British J. Cancer 72: 1364-1372.
Lindmo, T. et al., 1990, Immunometric assay by flow cytometry using mixture of two particle types of different affinity, J. Immunol. Meth. 126 (2): 183-189.
Orcutt, KD et al., 2010, A modular IgG-scFv bispecific antibody topology, Protein Engineering Design & Selection 23 (4): 221-228.
Orcutt, KD et al., 2011, Engineering an antibody with picomolar affinity to DOTA chelates of multiple radionuclides for pretargeted radioimmunotherapy and imaging, Nucl. Med. Biol. 38 (2): 223-233.
O'Donoghue, JA et al., 2011, 124I-huA33 antibody uptake is driven by a33 antigen concentration in tissues from colorectal cancer patients imaged by immuno-pet.J. Nucl. Med. 52: 1878-1885.
Scott, AM et al., 2005, A phase I trial of humanized monoclonal antibody A33 in patients with colorectal carcinoma: biodistribution, pharmacokinetics, and quantitative tumor uptake, Clin. Cancer Res. 11 (13): 4810-4817.
Welt, S. et al., 1994, Phase I / II study of iodine 131-labeled monoclonal antibody A33 in patients with advanced colon cancer, J. Clin. Oncol. 12 (8): 1561-71.
Welt, S. et al., 2003, Phase I study of anticolon cancer humanized antibody A33, Clin. Cancer Res. 9: 1338-1346.

Claims (77)

  A bispecific antibody comprising a first antigen binding site based on humanized antibody A33 (huA33) and a second antigen binding site based on monoclonal antibody 2D12.5.   The bispecificity according to claim 1, wherein the first antigen binding site and / or the second antigen binding site is one or more polypeptide chains or comprises one or more polypeptide chains. antibody.   3. The bispecific antibody of claim 2, wherein the one or more polypeptide chains comprise a heavy chain CDR found in the sequence set forth in Table 8.   4. The bispecific antibody of claim 3, wherein the heavy chain CDRs are found in a humanized A33 antibody (huA33).   3. The bispecific antibody of claim 2, wherein the one or more polypeptide chains comprise a light chain CDR found in the sequence set forth in Table 8.   6. The bispecific antibody of claim 5, wherein the light chain CDR is found at huA33.   3. The bispecific antibody of claim 2, wherein the one or more polypeptide chains comprise heavy and light chain CDRs found in one or more sequences set forth in Table 8.   8. The bispecific antibody of claim 7, wherein the heavy and light chain CDRs are found at huA33.   9. The bispecific antibody of any one of claims 1-8, wherein the bispecific antibody comprises SEQ ID NO: 2.   10. The bispecific antibody of claim 9, wherein the bispecific antibody further comprises SEQ ID NO: 6 or SEQ ID NO: 7.   2. The bispecific antibody of claim 1, wherein the first and / or second antigen binding site is a single chain variable fragment (scFv) or comprises an scFv.   The bispecific antibody according to claim 1, wherein the first antigen-binding site is composed of an immunoglobulin molecule, and the second antigen-binding site is composed of scFv, scFab, Fab or Fv.   13. The bispecific antibody of claim 12, wherein the second antigen binding site is scFv.   14. The bispecific antibody of claim 13, wherein the second antigen binding site is C825 scFv.   15. The bispecific antibody of claim 14, wherein C825 scFv is humanized.   16. A bispecific antibody according to claim 14 or 15, wherein scFv is linked to the C-terminus of the heavy chain of an immunoglobulin molecule.   16. The bispecific antibody according to claim 14 or 15, wherein scFv is linked to the C-terminus of the light chain of the immunoglobulin molecule.   18. The bispecific antibody of claim 17, wherein the bispecific antibody comprises SEQ ID NO: 2.   19. The bispecific antibody of claim 18, wherein the bispecific antibody further comprises SEQ ID NO: 6 or SEQ ID NO: 7.   21. An isolated nucleic acid molecule comprising a coding sequence for part or all of the polypeptide chain of a bispecific antibody according to any one of claims 1-19.   21. The isolated nucleic acid molecule of claim 20, wherein the coding sequence is codon optimized.   An expression vector comprising the nucleic acid sequence according to claim 20 or 21.   A host cell comprising the expression vector according to claim 22. A method for producing the bispecific antibody of any one of claims 1-19, comprising:
Culturing the host cell of claim 23 in a culture medium under conditions that allow expression of the bispecific antibody, and
Separating the bispecific antibody from the culture medium;
Said method.   20. A composition comprising the bispecific antibody of any one of claims 1-19.   26. A pharmaceutical composition comprising the composition of claim 25 or the bispecific antibody of any one of claims 1-19.   27. Use of the pharmaceutical composition according to claim 26 or the composition according to claim 25 for the treatment or diagnosis of cancer.   A method of treating a medical condition in a subject animal, wherein the medical condition is characterized by A33 expression, wherein the therapeutically effective amount of the bispecific antibody of any one of claims 1-19 is The method comprising the step of administering to a subject animal.   30. The method of claim 28, wherein the medical condition comprises an A33 positive tumor.   30. The method of claim 29, wherein the medical condition is colorectal cancer, gastric cancer or pancreatic cancer.   Treatment or detection of a symptom associated with A33 expression of a bispecific antibody according to any one of claims 1-19, a composition according to claim 25, or a pharmaceutical composition according to claim 26. Use for.   A method for killing tumor cells comprising the step of contacting tumor cells with a bispecific antibody, wherein the bispecific antibody is a first antigen binding based on humanized antibody A33 (huA33) Site and a second antigen binding site that binds to benzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA-Bn), the contact being a tumor The method, wherein the method is performed under conditions where cell killing is observed and for a time sufficient to kill the tumor cells.   35. The method of claim 32, wherein the contacting step comprises administering a bispecific antibody to the organism, wherein the administration is performed such that unbound bispecific antibody is removed from the blood.   The administering step comprises administering the bispecific antibody in combination with a conjugate comprising DOTA-Bn complexed with the payload, wherein the administration is performed such that the payload is delivered to the tumor cells. 34. A method according to claim 32 or claim 33.   34. The method of claim 32 or 33, wherein the administration is performed such that the tumor cells are substantially saturated with the bispecific antibody.   36. The method of claim 32, 33 or 35, wherein the administration is performed over a period of hours or days.   36. The method of claim 35, wherein the administration is performed such that the tumor cells are substantially saturated with the bispecific antibody prior to administration of the conjugate.   38. The method of claim 37, wherein the conjugate is administered over a period of minutes.   38. The administration is performed according to a combination regimen characterized by a therapeutic index of the conjugate, wherein the conjugate is at least 10 times better than the therapeutic index observed with a reference regimen where the conjugate is administered as a monotherapy. Or the method of 38.   Administration includes a first administration step in which a bispecific antibody is administered, a second administration step in which the conjugate is administered, and a different DOTA-Bn conjugate complexed with the same conjugate or payload. 38. A method according to claim 34 or 37, according to a regimen comprising one or more cycles of at least one third administration step.   41. The method of claim 40, wherein the regimen comprises 2, 3, 4, 5 or 6 cycles or more.   42. The method of claim 41, wherein the regimen achieves a curative result.   41. The method of claim 40, wherein the first administration step is performed such that the tumor cells are substantially saturated with the bispecific antibody.   44. The method of claim 40, 41 or 43, wherein the first administration step is performed before the second step and before any third administration step, and no further administration of the bispecific antibody is performed within the cycle. the method of.   45. The method of claim 44, wherein any cycle after the first cycle does not include administration of the bispecific antibody.   A method of inhibiting tumor growth comprising the step of contacting tumor cells with a bispecific antibody, said bispecific antibody comprising a first antigen binding site based on humanized antibody A33 and benzyl- Consists of a second antigen binding site that binds 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), the contact comprising DOTA- Said method is carried out under conditions and for a time sufficient for unbound bispecific antibodies to be removed from the blood in order to allow the final step of Bn killing tumor cells.   47. A method according to any one of claims 32-46, wherein the second antigen binding site is based on monoclonal antibody 2D12.5.   48. The method of claim 47, wherein the second antigen binding site is based on humanized 2D12.5.   49. The method of any one of claims 32-48, wherein the first and second antigen binding sites are scFvs.   49. The method of any one of claims 32-48, wherein the first antigen binding site is comprised of an immunoglobulin molecule and the second antigen binding site is comprised of an scFv.   51. The method of claim 50, wherein the second antigen binding site is C825 scFv.   52. The method of claim 51, wherein the C825 scFv is humanized.   53. The method of claim 51 or 52, wherein the scFv is linked to the immunoglobulin molecule at the C-terminus of the heavy chain.   52. The method of claim 51, wherein the scFv is linked to an immunoglobulin molecule at the C-terminus of the light chain.   55. The method of any one of claims 50-54, wherein the immunoglobulin molecule is IgG.   From immunoglobulin molecules based on humanized antibody A33 (huA33) and scFv that binds to benzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA-Bn) A bispecific antibody comprising the scFv based on C825 and linked to the C-terminus of the light chain of huA33, and the bispecific antibody is immunological when administered to an organism. Said bispecific antibody, characterized in that it has a limited effect.   57. The bispecific antibody of claim 56, wherein scFv is based on humanized C825.   58. The bispecific antibody of claim 56 or 57, wherein the immunoglobulin molecule is IgG.   59. The bispecific antibody of any one of claims 56 to 58, wherein the bispecific antibody comprises SEQ ID NO: 2.   60. The bispecific antibody of claim 59, wherein the bispecific antibody further comprises SEQ ID NO: 6 or SEQ ID NO: 7. A method of treating or diagnosing A33 positive cancer in a subject animal, comprising the following steps:
57. A step of administering to the subject animal the bispecific antibody of claim 56, wherein said administration comprises conditions and sufficient to localize said bispecific antibody to one or more tumors expressing A33 antigen. Performed in a short time,
Subsequently, the step of administering a removing agent to the subject animal, wherein the removing agent removes unbound bispecific antibody,
Subsequently, a step of administering radiolabeled DOTA-Bn to the subject animal.   62. The method of claim 61, further comprising the step of administering the bispecific antibody again to the subject animal.   64. The method of claim 62, wherein the step of re-administering the bispecific antibody to the subject animal is performed after administration of the remover.   64. The method of claim 63, wherein the step of re-administering the bispecific antibody to the subject animal is followed by re-administration of the remover to the subject animal.   65. The method of any one of claims 61-64, wherein the method does not substantially cause radiation damage to normal tissue.   66. The method of any one of claims 61-65, wherein the method results in an increase in therapeutic index of greater than 10 times.   67. The method according to any one of claims 61 to 66, wherein the removing agent is a dextran-based removing agent. 68. The method of any one of claims 61-67, wherein the radiolabeled DOTA is ¹⁷⁷ Lu-DOTA-Bn. Radiolabeled DOTA is ⁹⁰ Y-DOTA-Bn, The method according to any one of claims 61-67. Radiolabeled DOTA is ⁸⁶ Y-DOTA-Bn, The method according to any one of claims 61-67.   46. The method of any one of claims 34 and 37-45, wherein the payload is a toxic payload.   46. The method of any one of claims 34 and 37-45, wherein the payload is a biological response modifier.   73. A method according to claim 71 or 72, wherein the payload is selected from the group consisting of a detectable moiety and an active moiety.   The payload according to any one of claims 71-73, wherein the payload is or comprises a group selected from the group consisting of radioisotopes, peptides, nucleic acids, small molecules, nanoparticles, viruses and combinations thereof. The method according to item.   A method of killing tumor cells comprising multiple cycles of pre-targeted guided radioimmunotherapy using the bispecific antibody of claim 1.   76. The method of claim 75, comprising 3 cycles or more.   78. The method of claim 76, wherein the method achieves a curative result.